Magnetic Ionic Liquids, Methods of Making and Uses Thereof as Solvents in the Extraction and Preservation of Nucleic Acids

ABSTRACT

Described herein are methods for making and using magnetic ionic liquid that have at least one cationic component and at least one anionic component, where at least one of the cationic components or the anionic components is a paramagnetic component. The magnetic ionic liquids are capable of manipulation by an external magnetic field.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 15/048,265filed Feb. 19, 2016, now allowed, which claims priority to U.S.Provisional Application Ser. No. 62/118,901,filed under 35U.S.C. §111(b) on Feb. 20, 2015. The disclosures of all priority applicationsare incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant NumberCHE-1413199 awarded by the National Science Institute. The governmenthas certain rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Feb. 18, 2016, is named420_56859_SEQ_LIST_D2014-41.txt, and is 7,113 bytes in size.

[SEQ ID NO: 1] 5′-CAC CAT GAC AGT GGT CCC GGA GAA TTT CGT CCC-3′[SEQ ID NO: 2] 5′-ATG CCT ACA GTT ACT GAC TT-3′ [SEQ ID NO: 3]5′-TGC TGT TCC AGG GAC CT-3′ [SEQ ID NO: 4]5′-GAA TTC GGA TCC GGA CGC-3′

FIELD OF THE INVENTION

This disclosure describes magnetic ionic liquids having a cationiccomponent and an anionic component, where at least one of the cationiccomponents and the anionic components is a paramagnetic component. Themagnetic ionic liquid is capable of manipulation by an external magneticfield.

BACKGROUND OF THE INVENTION

Ionic liquids (ILs) are molten organic salts composed of organic cationsand inorganic/organic anions with low melting points (≤100° C.). Thesesolvents have garnered much attention due to their low vapor pressure atambient temperatures, high thermal stability, wide electrochemicalwindow, and multiple solvation capabilities.

The analysis of deoxyribonucleic acid (DNA) plays a central role in avariety of applications ranging from the determination of microbialdiversity in environmental samples, identifying pathogens in food,monitoring the levels of cell free nucleic acids in cancer prognosis(liquid biopsies), bioprospecting, and phylogenetic studies. Techniquessuch as polymerase chain reaction (PCR) and DNA sequencing enable theanalysis of extremely small quantities of DNA. However, the reliabilityand reproducibility of these methods largely depends on the quality ofthe sample. Complex environmental and biological samples often containcompounds such as humic acids, proteins, or lipids that can inhibit PCRamplification or DNA sequencing reactions. In these circumstances, aproper sample preparation technique is necessary to purify andpreconcentrate DNA for accurate and reproducible analysis.

Various sample preparation techniques have been developed for DNApurification including phenol-chloroform alkaline extraction, cesiumchloride-based density gradient ultracentrifugation, and solid phaseextraction (SPE) methods. Although useful in many cases, theseconventional DNA purification approaches often involve large samplevolumes, the use of organic solvents, time-consuming and laboriouscentrifugation steps, or multiple sample transfer steps that increasethe risk of contamination. DNA extraction based on commercial SPE kitsreduces the volume of organic solvent consumed as well as the timerequired for analysis, but the cost per sample remains high and thenumber of extractions that can be performed is limited. In some cases,the yield and purity of DNA obtained using different commercialextraction kits can be highly variable. Consequently, new methods thataddress the deficiencies of existing DNA sample preparation techniquesare particularly desirable.

SUMMARY OF THE INVENTION

Described herein are magnetic ionic liquids that have at least onecationic component and at least one anionic component. At least one ofthe cationic components or at least one of the anionic components is aparamagnetic component. The magnetic ionic liquid being capable ofmanipulation by an external magnetic field.

In certain embodiments, the cationic component is monocationic,dicationic or tricationic. Also, n certain embodiments, the cationiccomponent is an asymmetric cationic component.

In certain embodiments, the magnetic ionic liquid comprises multiplemagnetic iron(III) centers. Also, in certain embodiments, theparamagnetic component comprises a high spin transition metal. In oneembodiment, the paramagnetic component comprises a high-spin d⁵iron(III) center. For example, in certain embodiments, wherein the ionicliquid has an effective magnetic moment of up to 11.76 Bohr magnetons.

In certain embodiments, the magnetic ionic liquid comprises one or moreof: benzyl substituents; dysprosium; a benzimidazolium cation; animidazolium cation.

In certain embodiments, the anionic component comprises three anions.

In certain embodiments, the anionic component is selected from: a[FeCl₃Br⁻] anion and a [NTf₂ ⁻] anion.

Also described herein is a method of increasing the effective magneticmoment of an ionic liquid, comprising: incorporating an additionaliron(III) center into an ionic liquid, and thereby increasing theeffective magnetic moment of the ionic liquid. In certain embodiments,the method comprises using one or more steps shown in Scheme 1a orScheme 1b; Scheme 2; Scheme 3; or Scheme 4.

The magnetic ionic liquids are useful in conducting gas chromatography,where the magnetic ionic liquid is used as a stationary phase.

The magnetic ionic liquids are useful in conducting nucleic acidextraction, where the magnetic ionic liquid is used as a solvent toextract a nucleic acid from an aqueous solution; and, optionally,subjecting the extracted nucleic acid to a polymerase chain reaction(PCR) process.

In certain embodiment, the nucleic acid comprises DNA, or a syntheticDNA.

In certain embodiments, the method comprises dispersive liquid-liquidmicroextraction.

In certain embodiments, the method is single droplet extraction, or adispersive droplet extraction.

In certain embodiments, the method comprises conducting solid-phasemicroextraction using the magnetic ionic liquid as sorbent coatingimmobilized on a solid support to extract DNA from a solution.

Various aspects of this invention will become apparent to those skilledin the art from the following detailed description of the preferredembodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains one or more drawings executed incolor and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the U.S. Patent and Trademark Office upon request andpayment of the necessary fees.

FIG. 1A: Chemical structures of the three classes of hydrophobicmagnetic ionic liquids (MILs): monocationic, dicationic and tricationic.

FIG. 1B-FIG. 1C: The thermal properties of the MILs were evaluated usingthermal gravimetric analysis (TGA) (FIG. 1C), and differential scanningcalorimetry (DSC) (FIG. 1B).

FIG. 2A: Scheme 1a, showing synthesis of monocationic and dicationichydrophobic magnetic ionic liquids 1, 2 and 3 (MILs).

FIG. 2b : Scheme 1b, showing synthesis of monocationic and dicationichydrophobic magnetic ionic liquids 6 and 7 (MILs).

FIG. 3: Scheme 2, showing synthesis of dicationic heterocation-basedhydrophobic magnetic ionic liquids (MILs).

FIG. 4: Scheme 3, showing synthesis of symmetrical tricationic alkylatedand thiaalkylated-based hydrophobic magnetic ionic liquids (MILs).

FIG. 5: Scheme 4, showing synthesis of unsymmetrical tricationicheterocation-based hydrophobic magnetic ionic liquids (MILs).

FIG. 6: Structures of three hydrophobic MILs: (13) [(C₁₆BnIM)₂Cl₁₂²⁺][NTf2-, FeCl₃Br—]; (14) [(C₈)₃BnN+]—[FeCl₃Br—], and (15)[P_(6,6,6,14+)][FeCl₄—].

FIG. 7: Effect of MIL volume on extraction efficiency of stDNA. Opensquares (□) denote the [P_(6,6,6,14+)][FeCl₄—] MIL, and diamonds (♦)represent the [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] MIL.

FIG. 8: Effect of aqueous solution pH on the extraction efficiency ofstDNA using MIL-based dispersive droplet extraction. Open squares (□)represent the [P_(6,6,6,14+)][FeCl₄—] MIL, while diamonds (♦) indicatethe [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] MIL.

FIG. 9: Comparison of stDNA extraction efficiencies for threehydrophobic MILs from both a neat solution and a matrix containing metalions (NaCl, KCl, CaCl₂.2H₂O, and MgCl₂.6H₂O). Open bars represent DNAextraction from a neat aqueous solution, while gray bars indicateextraction of DNA from a matrix containing metal ions.

FIGS. 10A-10C: Effect of hydrophobic MIL type, pH, and albumin on theextraction efficiency of 20 kbp stDNA: (FIG. 10A) [(C₁₆BnIM)₂C₁₂²⁺][NTf2-, FeCl₃Br—], (FIG. 10B) [P_(6,6,6,14+)][FeCl₄—], and (FIG. 10C)[(C₈)₃BnN+][FeCl₃Br—]. Diamonds (⋄) represent extraction efficiencyvalues of stDNA, and circles (∘) denote extraction efficiencies ofprotein.

FIG. 11: Electropherogram obtained from the sequencing of the MTAP geneamplified from pDNA extracted by the [(C⁸)₃BnN+][FeCl₃Br—] MIL (SEQ IDNO: 5).

FIG. 12: Electropherogram obtained from the sequencing of the MTAP geneamplified from standard pDNA (SEQ ID NO: 6).

FIG. 13: Agarose gel electrophoresis of the MTAP gene after PCRamplification from pDNA recovered from the [(C₈)₃BnN+][FeCl₃Br—] MILextraction phase. Lane 1: a 250-25,000 bp DNA ladder. Lane 2: PCRproducts from pDNA recovered by rapid immersion of the DNA-enrichedmicrodroplet in Tris-HCl. Lane 3: PCR products obtained from pDNArecovered by semi-exhaustive DNA recovery.

FIG. 14: SEM image showing the cross-section of the PIL sorbent coatingused for extracting pDNA from aqueous solution after 40 extractions.

FIG. 15: Amplicon obtained following the preconcentration of 6.7 kbppDNA from aqueous solution using PIL and commercial PA SPME fibers. Lane1: PCR product from a 2 μL aliquot of 20 ng/mL of pDNA solution. Lane 2:PCR product following extraction/preconcentration using PIL-basedsorbent coating. Lane 3: PCR product following extraction using PAsorbent coating. Extraction conditions: pDNA concentration: 20 ng/mL;total solution volume: 10 mL; desorption solution: 1 M NaCl in 20 mMTris HCl; desorption solution volume: 50 μL; pH 4.0; extraction time: 30mM; desorption time: 15 min.

FIG. 16: Effect of NaCl on desorption of 6.7 kbp pDNA containing MTAPgene (879 bp) from PIL-based sorbent coating. Lane 1: PCR productsobtained following extraction and desorption of PIL fiber in aqueoussolution containing 1 M NaCl and 20 mM Tris HCl. Lane 2: PCR productsobtained following extraction and desorption of PIL fiber in 20 mM TrisHCl solution. Extraction conditions: pDNA concentration: 20 ng/mL; totalsolution volume: 10 mL; pH 4.0; extraction time: 20 mM; desorption time:15 min. desorption solution volume: 50 μL.

FIG. 17: Effect of extraction time on the extraction of 6.7 kbp pDNAcontaining the MTAP gene (879 bp). Lane 1: 5 mM; Lane 2: 10 mM; Lane 3:15 mM; Lane 4: 30 mM. Lane 5: represents a 390 ng MTAP gene standard.Extraction conditions: pDNA concentration: 20 ng/mL; total solutionvolume: 10 mL; desorption solution: 1 M NaCl in 20 mM Tris HCl;desorption solution volume: 50 μL; pH 4.0; desorption time: 15 mM.

FIG. 18: Effect of aqueous solution pH on the intensity of the ampliconobtained following PIL-based SPME of 6.7 kbp pDNA and PCR amplificationof an 879 bp gene. Conditions: pDNA concentration: 20 ng/mL; totalsolution volume: 10 mL; desorption solution: 1 M NaCl in 20 mM Tris HCl;desorption solution volume: 50 μL; pH 4.0; extraction time: 20 mM;desorption time: 15 mM.

FIG. 19: Electropherogram from the sequencing of the MTAP gene amplifiedfrom pDNA extracted by the PIL sorbent coating (SEQ ID NO: 7).

FIG. 20: Electropherogram from the sequencing of the MTAP gene amplifiedfrom standard pDNA (SEQ ID NO: 6).

FIG. 21: PCR products obtained following PIL-based SPME of a 1:1 mixtureof E. coli cells transformed with pDNA containing either the 1275 bp K4gene or the 879 bp MTAP gene. Lane 1: PCR products following PIL-basedSPME. Lane 2: includes a DNA ladder. Conditions: total solution volume:10 mL; desorption solution: 1 M NaCl; 20 mM Tris HCl; desorptionsolution volume: 50 μL; pH 4.0; extraction time: 20 min; desorptiontime: 15 mM.

FIG. 22: Detection of pDNA from E. coli cells containing either the 1275bp K4 gene or the 879 bp MTAP gene using the PIL-based SPME method. Lane1: DNA ladder. Lane 2: PCR products obtained following the extraction ofan aqueous solution spiked with 1.36×10⁸ cells containing K4 and1.44×10⁶cells containing MTAP. Conditions: total solution volume: 10 mL;desorption solution: 1 M NaCl in 20 mM Tris HCl; desorption solutionvolume: 50 μL; pH 4.0; extraction time: 20 mM; desorption time: 15 min.

DETAILED DESCRIPTION OF THE INVENTION Definitions

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and equivalents thereof knownto those skilled in the art, and so forth. As well, the terms “a” (or“an”), “one or more” and “at least one” can be used interchangeablyherein. It is also to be noted that the terms “comprising,” “including,”and “having” can be used interchangeably. The expression “of any ofclaims XX-YY” (wherein XX and YY refer to claim numbers) is intended toprovide a multiple dependent claim in the alternative form, and in someembodiments is interchangeable with the expression “as in any one ofclaims XX-YY.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresent disclosure, the preferred methods and materials are nowdescribed.

Every formulation or combination of components described or exemplifiedherein can be used to practice the materials and methods disclosedherein, unless otherwise stated.

When a group of substituents is disclosed herein, it is understood thatall individual members of that group and all subgroups, including anyisomers and enantiomers of the group members, are disclosed separately.When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and subcombinations possibleof the group are intended to be individually included in the disclosure.It is intended that any one or more members of any Markush group orlisting provided in the specification can be excluded if desired. When acompound is described herein such that a particular isomer or enantiomerof the compound is not specified, for example, in a formula or in achemical name, that description is intended to include each isomers andenantiomer of the compound described individual or in any combination.Additionally, unless otherwise specified, all isotopic variants ofcompounds disclosed herein are intended to be encompassed by thedisclosure. For example, it will be understood that any one or morehydrogens in a molecule disclosed can be replaced with deuterium ortritium. Isotopic variants of a molecule are generally useful asstandards in assays for the molecule and in chemical and biologicalresearch related to the molecule or its use. Specific names of compoundsare intended to be exemplary, as it is known that one of ordinary skillin the art can name the same compounds differently.

Thus, as used herein, the term “alkyl” includes straight, branched andcyclic alkyl groups, having up to 10 carbon atoms. An analogousconvention applies to other generic terms such as “alkenyl,” “alkynyl,”and the like. Furthermore, as used herein, the terms “alkyl,” “alkenyl,”“alkynyl,” and the like encompass both substituted and unsubstitutedgroups. In certain embodiments, as used herein, “lower alkyl” is used toindicate those alkyl groups (cyclic, acyclic, substituted,unsubstituted, branched or unbranched) having 1-6 carbon atoms.Non-limiting examples of alkyl groups include n-propyl, isopropyl, andisobutyl groups.

Illustrative aliphatic groups thus include, but are not limited to, forexample, methyl, ethyl, n-propyl, isopropyl, cyclopropyl,—CH₂-cyclopropyl, allyl, n-butyl, secbutyl, isobutyl, tert-butyl,cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl, isopentyl,tert-pentyl, cyclopentyl, —CH₂-cyclopentyl-n, hexyl, sec-hexyl,cyclohexyl, —CH₂cyclohexyl moieties and the like, which again, can bearone or more substituents. Illustrative alkynyl groups include, but arenot limited to, for example propargyl.

“Aryl” refers to an unsaturated aromatic or heteroaromatic carbocyclicgroup of from 1 to 15 carbon atoms having a single ring (e.g. phenyl) ormultiple condensed rings (e.g., naphthyl or anthryl). Preferred arylsinclude substituted aromatic C6-12 carbocycle; unsubstituted aromaticC1-10 heterocycle; substituted aromatic C1-10 heterocycle; wherein whensubstituted, the substitution is —XR.

Aralkyl refers to an alkyl connected to an aryl.

Unless otherwise constrained by the definition for the aryl substituent,such aryl groups can optionally be substituted with from 1 to 3substituents selected from the group consisting of hydroxy, acyl, alkyl,alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy,substituted alkenyl, substituted alkynyl, amino, aminoacyl, aminocarboxyesters, alkaryl, aryl, aryloxy, carboxyl, carboxylalkyl, acylamino,cyano, halo, nitro, heteroaryl, heterocyclic, oxyacyl, oxyacylamino,thioalkoxy, substituted thioalkoxy, trihalomethyl, mono- anddi-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclicamino, and unsymmetric di-substituted amines having differentsubstituents selected from alkyl, substituted alkyl, aryl, heteroaryland heterocyclic, and the like.

“Halogen” refers to fluoro, chloro, bromo and iodo and preferably iseither chloro or bromo.

“Heterocycle” or “heterocyclic” refers to a saturated or unsaturatedgroup having a single ring or multiple condensed rings, from 1 to 8carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfuror oxygen within the ring.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 3 substituents selected from the group consisting of hydroxy, acyl,alkyl, alkoxy, alkenyl, alkynyl, substituted alkyl, substituted alkoxy,substituted alkenyl, substituted alkynyl, amino, aminoacyl, aminocarboxyesters, alkaryl, aryl, aryloxy, carboxyl, carboxylalkyl, aminoacyl,cyano, halo, nitro, heteroaryl, heterocyclic, oxyacyl, oxyacylamino,thioalkoxy, substituted thioalkoxy, trihalomethyl, mono- anddi-alkylamino, mono- and di-(substituted alkyl)amino, mono- anddi-arylamino, mono- and di-heteroarylamino, mono- and di-heterocyclicamino, and unsynumetric di-substituted amines having differentsubstituents selected from alkyl, substituted alkyl, aryl, heteroaryland heterocyclic, and the like. Such heterocyclic groups can have asingle ring or multiple condensed rings. Preferred saturatedheterocyclics include morpholino, piperidinyl, and the like; andpreferred unsaturated heterocycles include pyridyl and the like.

It will be appreciated by one of ordinary skill in the art thatasymmetric centers can exist in the compounds of the present disclosure.Thus, the compounds and pharmaceutical compositions thereof can be inthe form of an individual enantiomer, diastereomer or geometric isomer,or can be in the form of a mixture of stereoisomers. It is to beunderstood that the present disclosure encompasses all possible isomerssuch as geometric isomers, optical isomers, stereoisomers and tautomersbased on an asymmetric carbon, which can occur in the structures of thecompounds, and mixtures of such isomers and compositions comprisingthose compounds, and is not limited to the specific stereochemistryshown for the compounds disclosed in the present specification. It willbe further appreciated that the absolute stereochemistry of some of thecompounds recited in the Exemplification herein cannot have beendetermined, and that when a stereochemistry was assigned for thosecompounds it is meant to be tentative and to indicate that a set ofdiastereomers exists for those compounds and/or that a diastereomer wasisolated in pure form. Furthermore, it will be appreciated that certainof the compounds disclosed herein contain one or more double bonds andthese double bonds can be either Z or E, unless otherwise indicated. Incertain embodiments, the compounds of the present disclosure areenantiopure compounds. In certain other embodiments, mixtures ofstereoisomers or diastereomers are provided.

Furthermore, this disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. The term “stable,”as used herein, preferably refers to compounds which possess stabilitysufficient to allow manufacture and which maintain the integrity of thecompound for a sufficient period of time to be detected and preferablyfor a sufficient period of time to be useful for the purposes detailedherein.

GENERAL DESCRIPTION

Certain embodiments of the present invention are defined in the Examplesherein. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

EXAMPLES Example 1 Synthesis of Hydrophobic Magnetic Ionic Liquids

In an effort to demonstrate the hydrophobicity and magneticsusceptibility in MILs, novel hydrophobic MILs were synthesized.

FIG. 1A groups the MILs into the following three classes: monocationicammonium-based MILs (1-3), symmetrical/unsymmetrical dicationic MILswith heteroanions (4a, 4b, and 6-8), and symmetrical/unsymmetricaltricationic MILs (10-12). Each synthetic route undertaken offers aunique approach toward incorporating hydrophobicity and magneticsusceptibility to the MIL structure. By designing MILs with multipleparamagnetic iron(III) centers, high effective magnetic moments (μeff)were achieved. The tricationic MIL 12 exhibited an μeff of 11.76 Bohrmagnetons (μB), which is a very high μeff for a MIL. Moreover, the lowwater solubilities observed for the MILs (less than 0.25% (w/v)) areideal for employing these compounds in magnet-based aqueous biphasicsystems. The thermal properties of each MIL were evaluated using thermalgravimetric analysis (TGA) (FIG. 1C) and differential scanningcalorimetry (DSC) (FIG. 1B).

Materials and Measurements

Imidazole (99%), 2-methylimdazole (99%), benzimidazole (98%),trioctylamine (97%), benzyl bromide (98%), 3-methoxybenzene (98%),4-(tert-butyl)benzene (97%), octanethiol (≥98.5%),2.2-dimethoxy-2-phenylacetophenone (99%), lithiumbis(trifluoromethyl)sulfonylimide, and 1,12-dibromododecane (98%) werepurchased from Acros Organics (Morris Plains, N.J., USA).

Acetonitrile, chloroform, dichloromethane, methanol, and diethyl etherwere purchased from Fisher Scientific (Fair Lawn, N.J., USA). Deuteratedchloroform and dimethyl sulfoxide (DMSO) were obtained from CambridgeIsotope Laboratories (Andover, Mass., USA). The NMR solvents were usedas received without additional drying. Iron(III) chloride hexahydrate(FeCl₃.6H₂O) (97%), 1-bromohexadecane (99%), 1-bromododecane (99%),1,3,5-tris(bromomethyl)benzene (97%), and dimethyl sulfoxide werepurchased from Sigma-Aldrich (St. Louis, Mo., USA). All reagents wereused as received without any further purification.

¹H NMR and ¹³C NMR spectra were recorded using a Varian 400 MHz nuclearmagnetic resonance spectrometer. Chemical shifts reported herein arerelative to tetramethylsilane. Mass spectra were obtained using anEsquire-LC-MS/MS from Bruker Daltonics. Thermogravimetric analyses wereperformed using a TA Instruments TGA Q600 thermogravimetric analyzer.All samples were loaded in platinum pans and heated at a rate of 5° C.min-1 under nitrogen flow (50 mL min-1). Differential scanningcalorimetry (DSC) traces were obtained using a Diamond differentialscanning calorimeter from PerkinElmer. Magnetic susceptibilitymeasurements were determined using a magnetic susceptibility balance(MSB) from Johnson Matthey. The MSB was calibrated with CuSO₄.5H₂O andvalidated using the data for the [P_(6,6,6,14+)][FeCl₄—] MIL. Visibleabsorption spectra were obtained using a Thermo Scientific Evolution 300UV-vis spectrophotometer. Absorption spectra of MILs were collectedusing acetonitrile as the solvent.

Synthesis of Monocationic Hydrophobic MILs

As shown in FIG. 2—Scheme 1, the synthesis of MILs 1-3 involves thereaction of trioctylamine (1 mmol) with bromomethyl substituents(R—CH2Br; R=benzene, methoxybenzene, and tert-butylbenzene; 1.1 mmol) inchloroform (25 mL) for 72 h under reflux conditions. The solvent wasevaporated under reduced pressure followed by washing the crude compoundwith hexanes (4×25 mL) under sonication to remove the unreacted startingmaterials. The bromide salt was then dried under vacuum at 60° C. for 12h. Characterization of compounds 1a-3a was performed using ¹H NMR, ¹³CNMR, and ESI-MS. After confirming the purity of the intermediates,compounds 1a-3a were reacted with equimolar amounts of FeCl₃.6H₂O inmethanol at room temperature under nitrogen atmosphere for 4 h. Afterevaporation of solvent, the crude MIL was washed with an excess ofdeionized water to remove unreacted FeCl₃ from the final product. TheMILs 1-3 were then dried under vacuum at 80° C. for 48 h andcharacterized using visible spectrophotometry and elemental analysis.

Characterization of 1a

Yield 92%. ¹H NMR (400 MHz; CDCl₃; ppm): δ0.86 (t, JH—H=6.35 Hz, 9H);1.25 (m, 30H); 1.75 (m, 6H); 3.29 (t, JH—H=8.06 Hz, 6H); 4.92 (s, 2H);7.42 (m, 3H); 7.53 (m, 2H). ¹³C NMR (400 MHz; CDCl₃; ppm): δ14.31;22.81; 22.97; 26.61; 29.28; 31.84; 59.08; 63.51; 127.68; 129.60; 130.97;132.76.

Characterization of MIL 1

Yield 91%. A dark reddish-brown viscous liquid. Characteristic bands for[FeCl₃Br—] anion were observed at 534, 619, and 688 nm using visiblespectroscopy. Elem. Anal. Calcd (%) for C₃₁H₅₈BrCl₃FeN (686.91): C,54.21; H, 8.51; N, 2.04. Found: C, 53.67; H, 8.06; N, 1.56.

Characterization of 2a

Yield 93%. ¹H NMR (400 MHz; CDCl₃; ppm): δ0.89 (t, JH—H=6.35 Hz, 9H);1.25 (m, 30H); 1.75 (m, 6H); 3.33 (t, JH—H=8.42 Hz, 6H); 3.86 (s, 3H);4.89 (s, 2H); 7.22 (m, 3H); 7.35 (m, 1H). ¹³C NMR (400 MHz; CDCl₃; ppm);δ14.41; 22.90; 23.08; 26.72; 29.41; 31.93; 56.05; 59.28; 63.62; 116.71;118.41; 124.61; 129.05; 130.62; 160.61.

Characterization of MIL 2

Yield 91%. A dark reddish-brown viscous liquid. Visible spectroscopyshowed characteristic bands for the [FeCl₃Br—] at 534, 619, and 688 nm.Elem. Anal. Calcd (%) for C₃₂H₆₀BrCl₃FeNO (716.93): C, 53.61; H, 8.44;N, 1.95. Found: C, 54.41; H, 7.64; N, 1.31.

Characterization of 3a

Yield 89%. ¹H NMR (400 MHz; CDCl₃; ppm): δ0.88 (t, JH—H=6.23 Hz, 9H);1.32 (m, 39H); 1.75 (m, 6H); 3.30 (t, JH—H=8.06 Hz, 6H); 4.82 (s, 2H);7.44 (m, 4H). ¹³C NMR (400 MHz; CDCl₃; ppm): δ14.32; 22.81; 22.94;26.62; 29.30; 29.36; 31.36; 31.87; 58.86; 63.12; 124.33; 126.61; 132.38;154.50.

Characterization of MIL 3

Yield 95%. A dark reddish-brown viscous liquid. Characteristic bands for[FeCl₃Br—] anion were observed at 534, 619, and 688 nm using visiblespectroscopy. Elem. Anal. Calcd (%) for C₃₅H₆₆BrCl₃FeN (743.01): C,56.58; H, 8.95; N, 1.89. Found: C, 57.38; H, 8.35; N, 1.62.

Synthesis of Compound 5

Compound 5 was synthesized as shown in FIG. 2B—Scheme 1b. Benzimidazole(2.00 g, 17.0 mmol) and potassium hydroxide (2.01 g, 35.0 mmol) weredissolved in dimethyl sulfoxide (30 mL) and stirred for 6 h at roomtemperature. Then, 1,12-dibromododecane (2.80 g, 8.53 mmol) was addedand continuously stirred for 24 h. Water (30 mL) was added to thereaction mixture, and the contents were subsequently transferred to aseparatory funnel. The reaction mixture was then extracted withchloroform (4×40 mL). The organic phases were washed several times withwater until the aqueous phase was a neutral pH. The organic phases werethen collected and dried over anhydrous sodium sulfate. Afterevaporation of the solvent, compound 5 was dried at 70° C. under reducedpressure for 12 h.

Characterization of 5

Yield 80-83%. ¹H NMR (400 MHz; DMSO; ppm): δ1.14 (m, 16H); 1.75 (m, 4H);4.21 (t, JH—H=6.84 Hz, 4H); 7.18 (m, 2H); 7.23 (m, 2H); 7.58(m, 2H);7.65 (m, 2H); 8.22 (s, 2H). ¹³C NMR (400 MHz; DMSO; ppm): δ26.07; 28.47;28.83; 29.35; 44.04; 110.41; 110.41; 119.38; 119.45; 121.32; 122.19;133.75; 143.46; 144.04.

Synthesis of Benzimidazolium-Containing Dicationic Heteroanion-BasedMILs

The synthesis of MILs 6 and 7 is shown in FIG. 2B—Scheme 1b. To astirred solution of compound 5 (1.24 mmol) in acetonitrile (15 mL),hexadecyl/benzyl bromide (2.60 mmol) was added and stirred at reflux for48 h. The solvent was evaporated under reduced pressure, and the crudecompound 6a/7a was washed with an excess of diethyl ether (4×20 mL)using sonication. The dicationic bromide salt (6a/7a) was then dried at80° C. under reduced pressure for 6 h to remove the residual solventfrom the product. Compounds 6b and 7b were prepared by reacting 6a/7a (1mmol) with lithium bisRtrifluoromethylisulfonyllimide (2.2 mmol) inmethanol at room temperature for 24 h. After solvent evaporation, thecrude compounds were washed with an excess of water and the halideimpurities monitored by adding silver nitrate to the aqueous phase.

Compounds 6c and 7c were obtained by reacting 6b/7b (1 mmol) withFeCl₃.6H₂O (1.2 mmol) in methanol at room temperature under nitrogenatmosphere for 12 h. The solvent was evaporated and the compounds werewashed with water (4×10 mL) under sonication to remove unreacted ironchloride. Compounds 6c/7c were dried under vacuum at 80° C. for 16 h toremove residual water.

Heteroanion-based MILs (6 and 7) were synthesized by mixing 6c or 7c (1mmol) with 6b or 7b (1 mmol), respectively, in methanol for 6 h at roomtemperature.

Characterization of 6a

Yield 91%. ¹H NMR (400 MHz; CDCl₃; ppm): δ0.82 (t, JH—H=6.32 Hz, 6H);1.18 (m, 68H); 2.01 (m, 8H); 4.60 (m, 8H); 7.74 (m, 8H); 11.33 (s, 2H).¹³C NMR (400 MHz; CDCl₃; ppm): δ14.17; 22.71; 26.41; 29.68; 29.70;31.94; 47.71; 113.13; 113.32; 127.20; 131.29; 131.30; 142.51. ESI-MS:m/2z (+) 426.7.

Characterization of 6b

Yield 87%. ¹H NMR (400 MHz; CDCl₃; ppm): δ0.87 (t, JH—H=6H); 1.25 (m,68H); 1.98 (m, 8H); 4.48 (m, 8H); 7.74 (m, 8H); 9.42 (s, 2H). ¹³C NMR(400 MHz; CDCl₃; ppm): δ14.40, 22.96; 26.39; 26.69; 29.07; 29.74, 29.95;32.19; 48.05; 113.33; 113.60; 127.68; 127.76; 131.61; 141.09. ESI-MS:m/2z (+) 426.7; m/z (−) 279.1.

Characterization of MIL 6

Yield 91%. The presence of the paramagnetic anion, [FeCl₃Br—], wasconfirmed using visible spectroscopy. Elem. Anal. Calcd (%) forC₆₀H₁₀₀BrCl₃F₆FeN₅O₄S₂ (1375.7): C, 52.38; H, 7.33; N, 5.09. Found: C,53.28; H, 7.30; N, 5.06.

Characterization of 7a

Yield 92%. ¹H NMR (400 MHz; DMSO; ppm): δ1.21 (m, 16H); 1.92 (m, 4H);4.52 (t, JH—H=7.14 Hz, 4H); 5.80 (s, 4H); 7.39 (m, 6H); 7.52 (m, 4H);7.66 (m, 4H); 7.97 (m, 2H); 8.11 (m, 2H); 10.10 (s, 2H). ¹³C NMR (400MHz; DMSO; ppm): δ25.82; 28.45; 28.49; 28.94; 46.84; 49.83; 113.90;126.64; 128.23; 128.96; 131.29; 134.12; 142.38. ESI-MS: m/2z (+) 292.4.

Characterization of 7b

Yield 90%. ¹H NMR (400 MHz; CDCl₃; ppm): δ1.24 (m, 16H); 1.99 (m, 4H);4.48 (t, JH—H=7.32 Hz, 4H); 5.63 (s, 4H); 7.36 (m, 10H); 7.59 (m, 2H);7.62 (m, 4H); 7.76 (m, 2H); 9.44 (s, 2H). ¹³C NMR (400 MHz; CDCl₃; ppm):δ26.29; 28.67; 29.01; 29.26; 48.01; 51.56; 113.41; 113.92; 118.33;121.52; 121.67; 128.22; 129.57; 132.38; 140.90. ESI-MS: m/2z (+) 292.4;m/z (−) 279.1.

Characterization of MIL 7

Yield 88%. A dark brown viscous liquid. Characteristic bands for the[FeCl₃Br—] anion were observed at 534, 619, and 688 nm using visiblespectroscopy. Elem. Anal. Calcd (%) for C₄₂H₄₈BrCl₃F₆FeN₅O₄S₂ (1107.09):C, 45.57; H, 4.37; N, 6.33. Found: C, 45.93; H, 4.14; N, 6.35.

Synthesis of MIL 8

As shown in FIG. 3—Scheme 2, compound 8 was synthesized. Briefly, asolution of 1,12-dibromododecane (8.20 g, 25.0 mmol in 30 mL ofchloroform) was added dropwise using a syringe to a stirred solution ofN-benzylimidazole (1.00 g, 6.32 mmol) in chloroform (40 mL) for 2 h. Thereaction mixture was allowed to stir for 12 h under reflux. The crudecompound 8a was obtained by evaporating the solvent under reducedpressure and washed with excess of hexanes and ethyl acetate (1:1; 3×30mL) under sonication to remove the unreacted starting materials.Compound 8a was dried in vacuum at 70° C. for 16 h to remove theresidual solvents. Compound 8b was obtained by anion exchange of 8a withLiNTf2 in water at room temperature for 24 h. After drying, compound 8b(0.30 g, 0.43 mmol) was reacted with previously synthesized1-hexadecylbenzimidazole (0.153 g, 0.450 mmol) in acetonitrile underreflux for 48 h. The resulting crude compound 8c was washed with anexcess of diethyl ether under sonication and dried under reducedpressure at 70° C. for 24 h. Finally, 8c (1 mmol) was reacted withFeCl₃.6H₂O (1.1 mmol) in dichloromethane at room temperature for 6 h toyield crude compound 8. After evaporation of the solvent, compound 8 waswashed several times with water to remove the unreacted iron chlorideand dried under vacuum for 4 h at 70° C.

Characterization of 8b

Yield 65%. ¹H NMR (400 MHz; DMSO; ppm): δ1.23 (m, 16H); 1.78 (m, 4H);3.52 (t, JH—H=6.59 Hz, 2H); 4.16 (t, JH—H=6.96 Hz, 2H); 5.41 (s, 2H);7.41 (m, 5H); 7.81 (m, 2H); 9.29 (s, 1H). ^(13C) NMR (400 MHz; DMSO;ppm): δ26.17; 28.19; 28.79; 28.99; 29.49; 29.55; 29.90; 32.89; 35.95;49.64; 52.63; 123.28; 123.49; 128.87; 129.69; 136.79. ESI-MS: m/z (+)407.3.

Characterization of 8c

Yield 82%. ¹H NMR (400 MHz; DMSO; ppm): δ0.84 (t, JH—H=6.59 Hz, 3H);1.21 (m, 42H); 1.76 (m, 2H); 1.89 (m, 4H); 4.15 (t, JH—H=6.96 Hz, 2H);4.48 (t, JH—H=6.59 Hz, 4H); 5.41 (s, 2H); 7.40 (m, 5H); 7.70 (m, 2H);7.82 (m, 3H); 8.11 (m, 2H); 9.29 (s, 1H); 9.80 (s, 1H). ¹³C NMR (400MHz; DMSO; ppm): δ13.98; 22.13; 25.58; 25.72; 25.82; 28.43; 29.06;29.31; 31.32; 46.69; 48.89; 51.98; 113.74; 122.61; 122.80; 126.57;128.21; 129.01; 131.10; 134.48; 136.10; 142.04. ESI-MS: m/2z (+) 334.7;m/z (−) 279.1.

Characterization of MIL 8

Yield 91%. A dark brown viscous liquid. Characteristic bands for the[FeCl₃Br—] anion were observed at 534, 619, and 688 nm using visiblespectroscopy. Elem. Anal. Calcd (%) for C₄₇H₇₂BrCl₃F₆FeN₅O₄S₂ (1191.33):C, 47.38; H, 6.09; N, 5.88. Found: C, 47.85; H, 6.17; N, 6.07.

Synthesis of Compound 9

Briefly, imidazole (0.59 g, 8.68 mmol) and potassium hydroxide (2.09 9,36.96 mmol) were dissolved in DMSO (30 mL) and stirred for 12 h at roomtemperature. Compound 9 was then obtained by adding1,3,5-tris(bromomethyl)benzene (1 g, 2.80 mmol) to the reaction flaskand stirring for 12 h. Water (30 mL) was added and the reaction mixturesubsequently transferred to a separatory funnel and extracted withchloroform (4×30 mL). The organic phases were washed several times withwater and dried over anhydrous sodium sulfate. After evaporation of thesolvent under reduced pressure, compound 9 was dried at 70° C. for 6 h.

Characterization of 9

Yield 70-75%. ¹H NMR (400 MHz; CDCl₃; ppm): δ5.08 (s, 6H); 6.84 (m, 6H);7.12 (s, 3H); 7.52 (s, 3H). ¹³C NMR (400 MHz; CDCl₃; ppm): δ50.37;119.40; 125.77; 130.45; 137.61; 138.65.

Synthesis of Compound 10

As shown in FIG. 4—Scheme 3, compound 10a was synthesized from compound9. Compound 10 was prepared by mixing compound 10a (1 mmol) withFeCl₃.6H₂O (3.1 mmol) in methanol under nitrogen atmosphere at roomtemperature for 4 h. After evaporation of solvent, compound 10 waswashed with water (4×10 mL) and dried under vacuum at 60° C. for 12 h.

Characterization of 10a

Yield 87%. ¹H NMR (400 MHz; CDCl₃; ppm): δ0.88 (t, JH—H=6.59 Hz, 9H);1.26 (m, 54H); 1.89 (m, 6H); 4.22 (t, JH—H=7.32 Hz, 6H); 5.55 (s, 6H);7.08 (s, 3H); 8.62 (m, 6H); 10.41 (s, 3H). ¹³C NMR (400 MHz; CDCl₃;ppm): δ14.36; 22.91; 26.51; 29.19; 29.55; 29.59; 29.81; 32.12; 50.45;52.21; 121.46; 124.62; 132.12; 135.77; 136.58.

Characterization of MIL 10

Yield 94%. A dark brown viscous liquid. The [FeCl₃Br—] anion wascharacterized using visible spectroscopy showing characteristic bands at534, 619, and 688 nm. Elem. Anal. Calcd (%) for C₅₄H₉₀Br₃Cl₉Fe₃N₆(1549.66): C, 41.85; H, 5.85; N, 5.42. Found: C, 41.65; H, 6.22; N,5.82;

Synthesis of MIL 11

For the synthesis of MIL 11, shown in FIG. 4—Scheme 3, allyl bromide(0.590 g, 4.89 mmol) was added to a stirred solution of compound 9 (0.50g, 1.6 mmol) in acetonitrile (15 mL) at 40-50° C. and stirred for 48 h.After evaporating the solvent in vacuo, the residue was washed withhexanes (3×15 mL) and dried under vacuum at 60° C. for 6 h to yieldcompound 11. A mixture of lla (0.30 g, 0.44 mmol), 1-octanethiol (0.579g, 3.96 mmol), and 2,2-dimethoxy-2-phenylacetophenone (0.169 g, 0.659mmol) in 10 mL of methanol/dichloromethane (1:1) was then transferred toa quartz tube and stirred until homogeneity. The contents were thenexposed to UV radiation (254 nm) for 16 h. The solvent was evaporatedand the crude compound washed with hexanes (4×30 mL) under sonication.Removal of residual solvents under vacuum yielded compound 11b. Finally,11b (1 mmol) was mixed with FeCl₃.6H₂O (3.10 mmol) in methanol at roomtemperature under nitrogen atmosphere for 4 h to form compound 11.Methanol was evaporated, and MIL 11 was washed with deionized water anddried at 70° C. for 12 h.

Characterization of 11a

Yield 91%; ¹H NMR (400 MHz; CDCl₃; ppm): δ4.89 (d, JH—H=6.59 Hz, 6H);5.45 (m, 3H); 5.48 (d, JH—H=4.76 Hz, 3H); 5.58 (s, 6H); 5.99 (m, 3H);7.16 (s, 3H); 8.46 (s, 3H); 8.52 (s, 3H); 10.34 (s, 3H). ¹³C NMR (400MHz; CDCl₃; ppm): δ52.28; 52.50; 121.42; 123.23; 124.58; 129.68; 131.91;135.71; 136.74.

Characterization of 11b

Yield 85%. ¹H NMR (400 MHz; CDCl₃; ppm): δ0.88 (t, JH—H=6.23 Hz, 9H);1.27 (m, 35H); 1.55 (m, 6H); 2.21 (m, 6H); 2.49 (t, JH—H=7.32 Hz, 6H);2.55 (t, JH—H=6.59 Hz, 6H); 4.39 (t, JH—H=6.96 Hz, 6H); 5.56 (s, 6H);7.17 (s, 3H); 8.58 (m, 6H); 10.42 (s, 3H). ¹³C NMR (400 MHz; CDCl₃;ppm): δ14.67; 23.20; 28.82; 29.43; 29.74; 30.03; 32.35; 32.71; 49.18;52.66; 122.25; 124.88; 132.53; 136.02; 137.15.

Characterization of MIL 11

Yield 91%. A dark brown viscous liquid. Characteristic bands for the[FeCl₃Br—] anion were observed at 534, 619, and 688 nm using visiblespectroscopy. Elem. Anal. Calcd (%) for C₅₁H₈₇Br₃Cl₉Fe₃N₆S₃ (1606.8): C,38.12; H, 5.46; N, 5.23. Found: C, 39.10; H, 5.62; N, 5.44.

Synthesis of MIL 12

The synthesis of MIL 12 is shown in FIG. 5—Scheme 4. Compound 9 (0.60 g,1.9 mmol) was dissolved in acetonitrile (50 mL) and added dropwise to asolution of bromobutane (0.517 g, 3.78 mmol) in acetonitrile (20 mL).The reaction mixture was stirred under reflux for 72 h. Afterevaporation of solvent, the crude compound was washed with diethyl ether(3×30 mL) under sonication for 30 min. Compound 12a (1 mmol) was thendissolved in water and allowed to react with LiNTf2 (2.20 mmol) at roomtemperature for 24 h. Compound 12b was then filtered and washed severaltimes with water and monitored for the presence of halide impurities byadding silver nitrate. Compound 12b (0.4 g, 0.4 mmol) was reacted withbromohexadecane (0.126 g, 0.413 mmol) in acetonitrile under reflux for48 h to obtain 12c. After solvent evaporation in vacuo, the residue waswashed several times with hexanes (4×10 mL) and dried at 70° C. for 12h. Finally, MIL 12 was synthesized by reacting 12c (0.30 g, 0.23 mmol)with FeCl₃.6H₂O (0.062 g, 0.25 mmol) in dichloromethane at roomtemperature for 4 h under nitrogen atmosphere. The solvent wasevaporated and MIL 12 was washed with water (10 mL) under sonication toremove unreacted iron chloride.

Characterization of 12a

Yield 78%. ¹NMR (400 MHz; DMSO; ppm): δ0.91 (t, JH—H=6.59 Hz, 6H); 1.26(m, 4H); 1.79 (m, 4H); 4.20 (m, 4H); 5.20 (m, 2H); 5.44 (m, 4H); 6.92(s, 1H); 7.32 (m, 3H); 7.75 (m, 6H); 9.42 (m, 2H). ¹³C NMR (400 MHz;DMSO; ppm): δ14.01; 19.51; 31.96; 49.41; 52.09; 123.27; 123.51; 128.35;129.36; 136.80; 136.93; 139.77; 140.08.

Characterization of 12b

Yield 91%. ¹H NMR (400 MHz; DMSO; ppm): δ0.91 (t, JH—H=6.59 Hz, 6H);1.23 (m, 4H); 1.77 (m, 4H); 4.17 (m, 4H); 5.22 (m, 2H); 5.41 (m, 4H);7.00 (s, 1H); 7.25 (m, 3H); 7.42 (m, 1H); 7.84 (m, 5H); 9.25 (s, 2H).¹³C NMR (400 MHz; DMSO; ppm): δ13.35; 18.87; 31.35; 48.79; 49.50; 51.51;122.60; 122.65; 122.92; 126.65; 127.65; 136.18; 136.26;

Characterization of 12c

Yield 85%. ¹H NMR (400 MHz; DMSO; ppm): δ0.91 (t, JH—H=6.59 Hz, 9H);1.23 (m, 28H); 1.78 (m, 6H); 4.18 (t, JH—H=7.32 Hz, 6H); 5.43 (s, 6H);7.46 (s, 3H); 7.76 (s, 3H); 7.85 (s, 3H); 9.33 (s, 3H). ¹³C NMR (400MHz; DMSO; ppm): δ13.34; 18.86; 22.14; 29.09; 31.33; 48.78; 51.41;122.58; 122.82; 128.66; 136.26.

Characterization of MIL 12

Yield 90%. A dark brown viscous liquid. The [FeCl₃Br—] anion wascharacterized using visible spectroscopy showing absorption bands at534, 619, and 688 nm. Elem. Anal. Calcd (%) for C₄₆H₆₉BrCl₃F₁₂FeN₈O₈S₄(1460.43): C, 37.83; H, 4.76; N, 7.67. Found: C, 37.33; H, 4.37; N,7.39.

Preparation of Monocationic Ammonium-Based Hydrophobic MILs

Monocationic MILs that lack acidic protons and are functionalized weredeveloped.

To improve the hydrophobic character of the resulting MIL, trioctylaminewas used and quaternized with alkyl halides. The reaction oftrioctylamine and butyl/decyl bromide resulted in low yields of thebromide salt (<20%) with most of the starting materials left unreacted,even after 7 days (based on 1H NMR spectroscopy). In contrast, the samereaction conditions with benzyl bromide produced higher yields (>90%)and proceeded to completion within 72 h, as shown in FIG. 2—Scheme 1.While not wishing to be bound by theory, it is now believed herein thatthis is due to resonance stabilization of the carbocation by thearomatic moiety. However, functionalized benzyl substituents including3-methoxybenzyl and 4-tert-butylbenzyl can be incorporated into thequaternary ammonium structure, which now allows for unique substitutionswithin the monocationic MIL framework.

The solubility of the ammonium-based MILs in water and hexane is shownin Table 1. Independent of the substituent functional groups imparted bythe quaternization reaction, MILs 1-3 were immiscible with water atcompositions as low as 0.1% (w/v) MIL. The hydrophobic properties of themonocationic MILs are thus useful for applications such as aqueousliquidliquid microextractions, where extremely low phase ratios ofextraction solvent are employed.

TABLE 1 Physicochemical and Magnetic Properties of Hydrophobic MILsSynthesized melting thermal solubility solubility MW point μeff^(b)stability^(c) in in MIL (g/mol) (° C.)^(a) (μB) (° C.) hexanes water  1686.9 −53.2 (Tg) 5.26 258 I I^(d)  2 716.9 <−65 5.60 203 I I^(d)  3743.01 −50.7 (Tg) 5.68 222 I I^(d)   4a 1035.0 −32.6 (Tg) 5.40 310 II^(e)   4b 1303.6 <25 5.37 294 I I^(e)   6c 1338.9 <25 7.58 299 I I^(e) 6 1375.7 −0.6 5.69 314 I Id  7 1107.1 −6.7 (Tg) 5.45 311 I I^(d)  81191.3 −16.7 5.30 312 I I^(d) 10 1549.6 <−65 11.25 312 I I^(e) 11 1606.8<−65 11.76 225 I I^(e) 12 1460.4 5.0 5.10 276 I I^(d) ^(a)-Tg = glasstransition temperature. ^(b)- μeff = effective magnetic moment measuredat 295 K. ^(c)-Thermal gravimetric analysis (TGA) = temperature at which5 wt % loss of MIL is observed; I = insoluble. ^(d)-Insoluble at 0.1%(w/v). ^(e)-Insoluble at 0.25% (w/v).

Structural Tuning of Symmetrical/Unsymmetrical Dicationic HydrophobicMILs

Dicationic ILs provide more opportunities for control of physical andchemical properties, as compared to conventional monocationic ILs. Thedicationic IL platform is especially useful for MILs, which require atleast a paramagnetic anion to provide sufficient magneticsusceptibility.

One cation in a dicationic system was paired with the [NTf2-] anion andproduced functionalized hydrophobic MILs based on imidazolium cations.In general, the synthesis of symmetrical imidazolium-based dicationicMILs was performed in three steps. First, a dicationic bromide salt wassynthesized with alkyl/aromatic substituents on the cation. A dodecyllinkage chain between the imidazolium-based cations was employed toincrease the conformational degrees of freedom, thus improving thelikelihood of forming low-melting MILs. Two homoanion precursors werethen synthesized from the dibromide salt, either by anion exchange toincorporate hydrophobic [NTf2-] anions or reaction with FeCl₃.6H₂O togenerate paramagnetic [FeCl₃Br—] anions. Finally, mixing equimolarquantities of the [NTf2-] and [FeCl₃Br—] salts produced dicationic MILswith heteroanions.

When functionalized with benzyl substituents, the imidazolium-baseddicationic MIL 4a exhibited water solubility below 0.25% (w/v), as shownin Table 1. Blocking the acidic C-2 proton of the hexadecylfunctionalized imidazolium dication with a methyl group (4b) resulted insimilar water solubility to 4a. The hydrophobicity of this class of MILswas significantly improved by replacing the imidazolium cation with thebenzimidazolium cation.

MILs 6 and 7 were synthesized according to FIG. 1—Scheme 1. Improvedhydrophobic character was observed for 6 and 7, which were insoluble inwater at 0.1% (w/v) MIL.

Another structural feature that affects the physicochemical propertiesof ILs is the presence of asymmetry in the molecule. Dicationic ILs areuniquely amenable to unsymmetrical archetypes since it is possible toindependently functionalize the cationic moieties. Since there wasimproved hydrophobicity obtained for dicationic MILs 6 and 7, a MIL thattethers benzimidazolium and imidazolium cations was produced. FIG.3—Scheme 2 illustrates the synthesis of the heterocationic MIL 8.Initially, reaction of 1,12-dibromododecane with benzylimidazole in a1:1 mole ratio resulted in formation of 20% (based on ¹H NMR) of thedibromide salt. However, increasing the mole ratio of1,12-dibromododecane to 4.5:1 significantly reduced the formation of thedibromide salt to ≤5%, based on ¹H NMR. The metathesis reaction ofcompound 8a with 1.2 mol equiv of LiNTf2 formed a precipitate (8b) whilethe dicationic [Br]/[NTf21] analogue remained in the aqueous phase.After the incorporation of the hydrophobic anion, compound 8b wasreacted with previously synthesized hexadecylbenzimidazole to generatethe unsymmetrical dicationic [Br—]/[NTf2-] salt 8c. Finally, hydrophobicMIL 8 was synthesized by reacting compound 8c with FeCl₃.6H₂O. Similarto MILs 1-3, 6, and 7, the heterocationic MIL 8 was insoluble in waterdown to 0.1% (w/v) MIL.

Synthesis of Symmetrical Alkylated/Thiaalkylated and UnsymmetricalTricationic Hydrophobic MILs

The synthesis of symmetrical tricationic hydrophobic MILs 10 and 11 isshown in FIG. 4—Scheme 3. With the aim of minimizing water solubility ofthe resulting MILs, a relatively hydrophobic benzene core functionalizedwith three imidazole moieties was selected as a precursor and preparedaccordingly. Compound 9 was then alkylated by reaction with1-bromododecane for 5 days in acetonitrile to generate compound 10a.Subsequent mixing with FeCl₃.6H₂O produced hydrophobic MIL 10. From FIG.4—Scheme 3, it is now shown that synthesis of the tribromide salt 10awas the most time-consuming step. In an effort to reduce the timerequired for the generation of a MIL with similar magneticsusceptibility and hydrophobicity to MIL 10, thiolene click chemistrywas employed.

The tricationic hydrophobic MIL 11 was synthesized as shown in FIG.4—Scheme 3. For synthesis of MIL 11, intermediate 9 was reacted withallyl bromide for 48 h in acetonitrile to produce compound 11a. Incomparison to the sluggish formation of the bromide salt 10a, compound11a was more rapidly generated, due to the resonance stabilization ofallylic carbocation. Compound 11b was prepared by reacting 11a withoctanethiol in a methanol/dichloromethane (1:1) solvent mixture in thepresence of UV irradiation for 16 h. Exclusive formation ofanti-Markovnikov-oriented products was confirmed by ¹H NMR. Excessamounts of unreacted starting materials were removed by washing thecrude compound with hexanes. Thus, the total reaction time required forthe generation of the thiaalkyl-based tribromide salt (11b) wassignificantly less in comparison with 10a. Finally, the hydrophobic MIL11 was prepared by reacting 11b with FeCl₃.6H₂O. Table 1 shows that MILs10 and 11 are insoluble in aqueous solution down to 0.25% (w/v) MIL.

In order to improve the hydrophobic character of the tricationic MILs,hydrophobic [NTf2] anions were incorporated into the molecularstructure, as shown in MIL 12. When intermediate 9 is reacted with alkylhalides such as 1-bromooctane, subsequent anion exchange with [NTf2]leads to the formation of room-temperature solids (Tm=51.1° C.).Formation of MILs with lower melting points involved reactingintermediate 9 with 1-bromobutane to yield the dibromide salt (12a), asshown in FIG. 5—Scheme 4. Hydrophobic [NTf2-] anions were thenincorporated by metathesis reaction of 12a. Generation of theunsymmetrical tricationic IL was accomplished by reacting 12b with1-bromohexadecane to produce compound 12c. Finally, MIL 12 was generatedby reacting compound 12c with FeCl₃.6H₂O in dichloromethane for 4 h. Asshown in Table 1, MIL 12 exhibited improved hydrophobicity in comparisonto MILs 10 and 11.

Thermal Properties of Hydrophobic MILs

The phase transition behavior and thermal stability of the hydrophobicMILs were examined using DSC and TGA, and the results compiled inTable 1. Monocationic MILs 1 and 3 exhibited glass transitiontemperatures (Tg) of −53.2 and −50.7° C., respectively. A melting point(Tm) for MIL 2 could not be detected above −65° C. These values weremuch lower in comparison with ammonium-based MILs possessing only linearalkyl substituents. The total number of carbon atoms and asymmetry ofthe cation structure govern the Tm of quaternary ammonium salts. Hence,the relatively low Tg of MILs 1 and 3 may be explained by the asymmetryresulting from incorporation of aromatic moieties within the cationstructure. Interestingly, while MIL 1 was thermally stable up to 258°C., similar weight loss (5%) was observed at lower temperatures for themethoxy (2) and tert-butyl (3) functionalized monocationic MILs.

Symmetrical dicationic MILs with heteroanions (4a, 4b, 6, and 7)exhibited higher phase transition temperatures than the monocationicMILs but were nonetheless liquids at room temperature. Despite similarcation substituents, MIL 7 exhibited a higher Tg than 4a which may beattributed to enhanced 7C-7C interactions as a consequence of thebenzimidazolium cation. The dicationic MILs exhibited no more than 5%weight loss below temperatures ranging from 294 to 312° C. Nosignificant enhancement in thermal stability was observed for thedicationic MILs containing [NTf2-]/[FeCl₃Br—] heteroanions compared tothose containing 2[FeCl₃Br—] homoanions.

The effect of dissimilar cationic moieties on the melting point ofdicationic MILs is shown in FIG. 1B. Unsymmetrical replacement of thehexadecylbenzimidazolium cation with the benzylimidazolium cationlowered the Tm from −0.6 to −16.7° C. (MILs 6 and 8, respectively), anoutcome that is attributed to diminished π-π0 interactions as well asremoval of the dication symmetry component. Analogous to conventionaldicationic ILs, endothermic peaks in DSC traces were quite broad withobservable shouldering. This is now believed herein to be due to thehighly flexible nature of the MIL cations, which allows differentconformations and multiple step phase transitions.

Phase transitions were not observed above −65° C. for the heavilyalkylated/thiaalkylated symmetrical tricationic MILs 10 and 11. Bothcompounds 10 and 11 are room temperature liquids in contrast to theircorresponding bromide salts. This is a function of incorporating theweaker coordinating [FeCl₃Br—] anion. The thermal decompositiontemperatures varied significantly between the alkylated (10) andthiaalkylated (11) hydrophobic tricationic MILs. Possessing a morethermally labile C—S bond, MIL 11 exhibited 5% weight loss at 225° C.compared to 312 and 275° C. for MIL 10 and 12, respectively.

Magnetic Properties of Hydrophobic MILs

The paramagnetic properties of MILs provide a unique advantage overconventional ILs by permitting control over substrate motion through theapplication of an external magnetic field. MILs containing high-spin d5iron(III) centers are well characterized owing to the abundance and lowcost of iron materials and exhibit paramagnetism at ambienttemperatures. Table 1 shows the effective magnetic moments for all MILs,determined using an Evans magnetic susceptibility balance. The generalexpression for molar magnetic susceptibility using the Evans balance isshown in eq 1:

$\begin{matrix}{X_{M} = \frac{C_{bal}{{LM}\left( {R - R_{o}} \right)}}{10^{9}m}} & (1)\end{matrix}$

where XM represents the molar magnetic susceptibility, C_(bal) is thebalance calibration constant, L corresponds to the length of the samplein the tube, M is the molecular weight of the compound being measured, Ris the instrument reading for the sample in the tube, R_(o) is theinstrument reading for the empty sample tube, and m is the mass ofsample introduced into the Evan's balance. From XM, it is possible tocalculate μeff according to eq 2:

μ_(eff)=2.83√{square root over (X_(M)T)}  (2)

where T is the absolute temperature. The μeff for ammonium-basedmonocationic MILs ranged from 5.26 to 5.68 μBand were comparable tothose determined for heteroanionic MILs possessing a single [FeCl₃Br—]anion (4a, 4b, 6, 7, 8, and 12). In order to increase the magneticsusceptibility of MILs, multiple paramagnetic iron(III) centers wereincorporated into the MIL structure. For example, dicationic MIL 6c wasdesigned to possess two paramagnetic iron(III) centers providing anincrease in μeff to nearly 7.6 μB. Further enhancement of magneticsusceptibility was observed in tricationic MILs 10 and 11, whichexhibited μeff=11.25 μB and 11.76 μB, respectively. The iron(III)-basedMILs represent an inexpensive and useful alternative to MILs based onlanthanides, such as Dy(III) (μeff=10.6 μB). Moreover, the higherresponse of MILs 10 and 11 toward magnetic fields enables the use ofsmaller magnets with lower field strength for their manipulation insolution, which is useful in miniaturized magnet-based systems.

Discussion of Example 1

In Example 1, three general classes of hydrophobic MILs weresuccessfully synthesized and characterized. Within each class, thesynthetic approaches now described herein were used to control themagnetic and physicochemical properties of MILs. The incorporation ofbenzyl substituents within the MIL structure of monocationic quaternaryammonium-based hydrophobic MILs produced lower melting point compoundscompared to linear alkyl substituents. The hydrophobicity of dicationicMILs was enhanced by replacing imidazolium cations with benzimidazoliumcations, resulting in MILs that are insoluble in water down to 0.1%(w/v). Moreover, the inclusion of asymmetry within the cationic portionof dicationic MILs lowered the melting point without sacrificinghydrophobicity or magnetic susceptibility. Additionally, increasing thenumber of paramagnetic iron(III) centers in the MIL structure resultedin higher μeff values. Throughout the preparation of tricationic MILs, acommon intermediate was chosen and modified to alter the hydrophobicityand magnetic properties of resulting MILs. Tricationic MILs containingthree [FeCl₃Br—] anions exhibited μeff values as high as 11.76 μB,representing the highest known value reported for MILs. The synthesis ofiron(III)-based tricationic hydrophobic MILs possessing large μeff is aninexpensive alternative to MILs based on lanthanides.

Example 2 Extraction of DNA by Magnetic Ionic Liquids: Tunable Solventsfor Rapid and Selective DNA Analysis

Several hydrophobic MILs, namely1,12-bis[N-(N′-hexadecylbenzimidazolium) dodecanebis[(trifluoromethylisulfonyl]imide bromotrichloferrate(III)[(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—]) (13); benzyltrioctylammoniumbromotrichloroferrate(III) ([(C₈)₃BnN⁺][FeCl₃Br—]) (14); and,trihexyl(tetradecyl)phosphonium tetrachloroferrate(III)([P_(6,6,6,14+)][FeCl₄—]) (15), were used for the direct extraction ofDNA from an aqueous solution. Isolation of the extraction phase wasachieved by applying an external magnetic field, thereby circumventingtime-consuming centrifugation steps. The optimized MIL-based extractionprocedures are capable of performing rapid and highly efficientextraction of double-stranded and single-stranded DNA from a matrixcontaining metal ions and protein. Plasmid DNA (pDNA) extracted from abacterial cell lysate using the MIL-based method is now shown to be ahigh quality template for PCR.

Reagents

Benzimidazole, trioctylamine, 1,12-dibromododecane, and guanidinehydrochloride (GuHCl) were purchased from Acros Organics (NJ, USA).Trihexyl(tetradecyl)phosphonium chloride was purchased from StremChemicals (Newburyport, Mass., USA). Deuterated chloroform was obtainedthrough Cambridge Isotope Laboratories (Andover, Mass. USA). Iron(III)chloride hexahydrate (FeCl₃.6H₂O), 1-bromohexadecane, benzyl bromide,sodium dodecyl sulfate (SDS), albumin from chicken egg white, and DNAsodium salt from salmon testes (stDNA, approximately 20 kbp) werepurchased from Sigma-Aldrich (St. Louis, Mo., USA). Sodium chloride,sodium hydroxide, potassium chloride, calcium chloride dihydrate,magnesium chloride hexahydrate, potassium acetate, silica gel sorbent(230-400 mesh), and tris(hydroxymethyl)aminomethane hydrochloride(Tris-HCl) were purchased from Fisher Scientific (Fair Lawn, N.J., USA).Synthetic oligonucleotides including duplex (20 bp, molecular weight=12232 Da), single-stranded DNA oligonucleotides (33 mer, molecularweight=10 075 Da), and primers were purchased from IDT (Coralville,Iowa, USA). The pET-32 plasmid was obtained from EMD Millipore(Billerica, Mass., USA). NEB 5-alpha Competent Escherichia coli cellsand Phusion High-Fidelity DNA Polymerase were obtained from New EnglandBiolabs (Ipswich, Mass., USA). Agarose andtris(hydroxymethyl)aminomethane (Tris) were obtained from P212121(Ypsilanti, Mich., USA). A 1 Kb Plus DNA Ladder (250-25,000 bp) wasobtained from Gold Biotechnology, Inc. (St. Louis, Mo., USA) with SYBRSafe DNA gel stain and bromophenol blue being supplied by LifeTechnologies (Carlsbad, Calif., USA) and Santa Cruz Biotech (Dallas,Tex., USA), respectively. QIAquick Gel Extraction and QIAamp DNA MiniKits were purchased from QIAgen (Valencia, Calif., USA). Deionized water(18.2 MΩ cm) obtained from a Milli-Q water purification system was usedfor the preparation of all solutions (Millipore, Bedford, Mass., USA).

Synthesis and Characterization of Hydrophobic Magnetic Ionic Liquids

The synthesis of two hydrophobic MILs, namely, [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-,FeCl₃Br—] (13) and [(C₈)₃BnN+][FeCl₃Br—] (14), was carried out asdescribed herein. ¹H NMR, ¹³C NMR, ESI-MS, and UV-vis were used tocharacterize the three MILs.

Instrumentation

High performance liquid chromatography with UV detection was performedon a LC-20A liquid chromatograph (Shimadzu, Japan) consisting of twoLC-20AT pumps, a SPD-20 UV/vis detector, and a DGU-20A3 degasser.Chromatographic separations were performed on a 35 mm×4.6 mm i.d.×2.5 μmTSKgel DEAE-NPR anion exchange column with a 5 mm×4.6 mm i.d.×5 μmTSKgel DEAE-NPR guard column from Tosoh Bioscience (King of Prussia,Pa.). The column was equilibrated with a mobile phase composition of50:50 (A) 20 mM Tris-HCl (pH 8), and (B) 1 M NaCl/20 mM Tris-HCl (pH 8).For stDNA analysis, gradient elution was performed beginning with 50%mobile phase B and increased to 100% B over 10 min. In the separation ofssDNA as well as DNA and albumin, the column was first equilibrated with20 mM Tris-HCl followed by gradient elution from 0% to 50% B over 10 mMand then 50% to 100% B over 5 mM. A flow rate of 1 mL min-1 was used forall HPLC separations. DNA and albumin were detected at 260 and 280 nm,respectively.

All extractions were performed in 4 mL screw cap vials. Isolation of themagnetic ionic liquid extraction phase was achieved using a cylindermagnet (B=0.9 T) or rod magnet (B=0.66 T) obtained from K&J Magnetics(Pipersville, Pa.). A Techne FTgene2D thermal cycler (Burlington, N.J.,USA) was used for all PCR experiments. Agarose gel electrophoresis wasperformed in a Neo/Sci (Rochester, N.Y.) electrophoresis chamber with adual output power supply. Gels were visualized at 468 nm on a Pearl BlueTransilluminator (Pearl Biotech, San Francisco, Calif.).

MIL-Based Single Droplet Extraction

The MIL-based static single droplet extraction (SDE) method wasperformed. Briefly, a 20 μL droplet of MIL was suspended from a magneticrod (B=0.66 T) and lowered into a 4.17 nM solution of stDNA buffered by20 mM Tris-HCl (pH 8). After 5-120 min, the MIL droplet was removed fromthe sample and a portion of the aqueous phase subjected to HPLC analysisto determine the concentration of DNA remaining after extraction.

MIL-Based Dispersive Droplet Extraction

The general MIL-based dispersive droplet extraction (DDE) approach wasused. Briefly, a 4.17 nM solution of stDNA was prepared in 20 mMTris-HCl (pH 8). An optimized volume of MIL (typically 20 μL) was addedto the aqueous DNA solution and manually shaken for 5-60 s, resulting ina dispersion of the hydrophobic MIL in the aqueous phase. In the case ofthe [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] MIL, it was gently heated priorto extraction. The vial was then placed in a 0.9 T magnetic field tofacilitate the rapid isolation of MIL followed by HPLC analysis of a 20μL aliquot of the aqueous phase.

Extraction of Synthetic Oligonucleotides and Duplex DNA

Solutions of synthetic oligonucleotides and duplex DNA were preparedsuch that the mass of DNA in aqueous solution was consistent with theexperiments involving stDNA (100 μg of stDNA in 2 mL of Tris-HCl). Forextractions of ssDNA, a 33 base oligonucleotide with sequence 5′-CAC CATGAC AGT GGT CCC GGA GAA TTT CGT CCC-3′ [SEQ ID NO:1] was dissolved in 20mM Tris-HCl (pH 8) resulting in a final concentration of 1499 nM. In thecase of synthetic dsDNA, an aqueous solution containing 1224 nM of 20 bpduplex (sequence: 5′-ATG CCT ACA GTT ACT GAC TT-3′ [SEQ ID NO:2] and itscomplementary strand) was prepared in 20 mM Tris-HCl (pH 8). Solutionscontaining single-stranded oligonucleotides or duplex DNA were subjectedto MIL-based DDE with a 20 μL portion of the aqueous phase beinganalyzed by HPLC.

Extraction of DNA from a Complex Matrix

Sample matrices containing either metal ions or protein (albumin) wereprepared from stock solutions. A sample solution containing 388 mM NaCl,153 mM KCl, 38.1 mM CaCl₂.2H₂O, 28.3 mM MgCl₂.6H₂O, and 4.17 nM stDNAwas extracted in triplicate using MIL-based DDE for all three MILs. Forexperiments involving protein as a matrix component, the samples wereprepared at an albumin concentration of 3.4 μM and stDNA concentrationof 4.17 nM with the pH varied from 3.5 to 8.

PCR and DNA Sequence Analysis

For DNA sequence analysis, a modified pET-32 plasmid containing an 879bp gene encoding human 5′-methylthioadenosine phosphorylase (MTAP) wasextracted using the [(C₈)₃BnN+][FeCl₃Br—] MIL in the DDE approach. ThepDNA-enriched MIL microdroplet was removed from solution using a 0.66 Trod magnet and stored at room temperature for 24 h. Recovery of the pDNAwas achieved by dispersion of the MIL microdroplet in 200 μL of 20 mMTris-HCl (pH 8) for 2 min. A 2 μL aliquot of the aqueous phase wassubjected to PCR using primers for the MTAP gene. The PCR products wereseparated by agarose gel electrophoresis, and the band containing theMTAP gene was extracted from the gel using a QIAquick Gel ExtractionKit. An external DNA sequencing service (Eurofins Genomics, Huntsville,Ala.) performed sequence analysis of the MTAP gene amplified from thepDNA recovered from the MIL extraction phase.

Amplification of the MTAP gene was performed using the primers 5′-TGCTGT TCC AGG GAC CT-3′ [SEQ ID NO:3] (molecular weight=5,177.4 Da) and5′-GAA TTC GGA TCC GGA CGC-3′ [SEQ ID NO:4] (molecular weight=5,524.6Da). A 2 μL aliquot of aqueous solution containing pDNA recovered fromthe MIL extraction phase was added to a PCR tube with 34.5 μL of DI H₂Oand 10 μL of 5× Phusion HF buffer. Primers and dNTPs were added toachieve a final concentration of 0.2 μM and 200 μM, respectively.Finally, 1 unit of Phusion High Fidelity DNA polymerase was added to thereaction mixture. The total reaction volume was 50 μL. The followingtemperature program was used for amplification of MTAP: 5 mM initialdenaturation at 95° C. and 30 cycles comprised of a 30 s denaturationstep at 95° C., a 45 s hold at 54° C. for annealing, and a 45 selongation step at 72° C.

Recovery of DNA from the MIL Extraction Phase

Following MIL-based DDE of a 4.17 nM solution of stDNA, the DNA-enrichedMIL microdroplet was first transferred into a microcentrifuge tubecontaining 1 mL of 3 M potassium acetate (pH 4.8) and vortexed for 2min, ensuring a homogeneous solution. A silica sorbent column wasconstructed by measuring 750 mg of silica particles into a Pasteur pipetwith the exit end blocked by a glass wool frit. The column wasconditioned with 2 mL of 6 M GuHCl, and the sample was subsequentlyloaded at approximately 1 mL min-1. The sorbent was flushed with 1 mL ofisopropanol and the first fraction collected. Next, 750 μL of ethanolwas added, and the turbid solution was centrifuged at 16,200 g for 15mM. The pellet was washed with 80% ethanol for 1 mM. The sample wascentrifuged once more at 16,200 g for 10 mM, and the supernatant wasdecanted. The pellet was dried under an air stream and reconstituted in100 μL of Tris-HCl (pH 8), and a 20 μL aliquot was removed for HPLCanalysis.

As an alternative, a rapid approach to DNA recovery was employed. AfterMIL-based DDE, the DNA-enriched MIL microdroplet was collected fromaqueous solution using a 0.66 T rod magnet and immersed in 200 μL ofTris-HCl (pH 8) for 2 mM. The microdroplet was then removed fromsolution and the aliquot subjected to PCR amplification.

Extraction of DNA from Bacterial Cell Lysate

The conditions used to culture NEB 5-alpha Competent E. coli cellscontaining pDNA were as follows: NEB 5-alpha Competent E. coli cellswere transformed with modified pET-32 plasmid DNA (pDNA) containing the5′-methylthioadenosine phosphorylase (MTAP) gene. A 1 μL aliquot ofpurified pDNA (347 ng μL-1) was added to a microcentrifuge tubecontaining 20 μL of competent E. coli cells. The mixture was set on icefor 30 mM. The solution was placed in a water bath at 40° C. for 42 sand subsequently chilled on ice for 2 mM. A 250 μL aliquot of LuriaBertani (LB) media was added to the solution, which was then incubatedat 37° C. for 1 h. Transformed E. coli cells were incubated in 100 mL ofLB media with 200 μg mL-1 carbenicillin at 37° C. for 20 h.

A 10 mL aliquot of an overnight E. coli cell culture was centrifuged at16,200 g for 5 min and resuspended in 300 μL of 20 mM Tris buffercontaining 10 mM EDTA (pH 8). Lysozyme (200 μg) was added to thesolution, which was then incubated for 5 mM at room temperature,followed by the addition of 600 μL of 0.2 N NaOH, 1% SDS (w/v). Aftergentle mixing of the solution, 400 μL of 3 M potassium acetate (pH 4.8)was added. The contents were thoroughly mixed and centrifuged at 16,200g for 10 mM. A 400 μL aliquot of the supernatant was transferred to aclean vial, and the solution was extracted using the MIL-based DDEapproach. The pDNA was then recovered using either the aforementionedsilica-based or the rapid immersion procedure prior to PCRamplification.

Structural Design of Hydrophobic MILs for DNA Extraction

In order to develop sufficiently hydrophobic MILs that still possessparamagnetic behavior, a dicationic platform with [NTf2-]/[FeCl₃Br—]heteroanions was chosen. As shown in FIG. 6, the [(C₁₆BnIM)₂C₁₂²⁺][NTf2-, FeCl₃Br—] MIL is comprised of both hydrophobic andparamagnetic anions. Although a greater magnetic moment can be achievedby employing two [FeCl₃Br—] anions in a dicationic MIL, increasedwater-miscibility is also observed. The cationic portions of the[(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] and [(C₈)₃BnN+][FeCl₃Br—] MILs arefunctionalized with long alkyl chains and benzyl moieties, whichsignificantly increases their overall hydrophobicity.

Optimization of DNA Extraction Mode

The amount of DNA extracted by the hydrophobic MIL extraction phases wasevaluated indirectly by subjecting an aliquot of the postextractionaqueous phase to HPLC analysis. An external calibration curve for bothdsDNA and ssDNA was established and used to calculate the DNAconcentration in aqueous solution. Values of extraction efficiency (E)were obtained using the relationship between the DNA concentration inthe aqueous phase following extraction (C_(aq)) and the concentration ofDNA in the standard solution (C_(st)), as shown in eq 3:

$\begin{matrix}{E = {\left\lbrack {1 - \frac{C_{aq}}{C_{st}}} \right\rbrack \times 100}} & (3)\end{matrix}$

Time-consuming centrifugation steps in extraction and purificationprotocols represent a major bottleneck in nucleic acid samplepreparation. In the development of MIL-based DNA extraction methods,considerable attention was given to the compromise between extractiontime and efficiency. Identical volumes of MIL were used to extract DNAfrom an aqueous solution using both SDE and DDE modes. One advantage ofDDE over SDE is the dynamic mixing of the MIL extraction solvent withthe aqueous medium, which allows for rapid distribution of DNA betweenthe two phases. This is illustrated in Table 2 where the extractionefficiency of stDNA is shown for the [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—]MIL using both SDE and DDE modes. The relatively low extractionefficiencies observed for the SDE technique, particularly at shortextraction times, are likely due to less available MIL surface area forinteraction with DNA when compared to DDE. The precision of eachextraction mode ranged from 1.6 to 8.7% and 0.4 to 3.4% for SDE and DDE,respectively, using triplicate extractions. While the SDE mode required2 h to achieve an extraction efficiency of 63.1%, the DDE mode providedefficiencies greater than 76% after just 30 s of dynamic mixing and 5 sof phase isolation by exposure to a magnetic field. No appreciable gainin extraction efficiency was observed when the magnetic field wasapplied at time points greater than 5 s. Therefore, DDE was selected asthe optimum extraction mode for subsequent DNA extractions using thethree hydrophobic MILs.

TABLE 2 Comparison of Single Droplet and Dispersive Droplet ExtractionModes for the Extraction of stDNA from an Aqueous Solution Using the[(C₁₆BnIM)₂C₁₂ ²⁺][NTf2−, FeCl₃Br−] MIL Single Droplet Extraction^(a) %extraction efficiency time (min) (n = 3) % RSD 10 5.5 1.6 20 33.3 3.0 3040.5 8.7 60 60.3 3.3 90 61.6 8.6 120 63.1 4.1 Dispersive DropletExtraction^(b) % extraction efficiency time (s)^(c) (n = 3) % RSD 5 76.83.4 30 75.6 0.4 60 79.3 2.3 120 76.5 2.1 300 77.0 1.2 ^(a)Conditions:DNA concentration: 4.17 nM; volume of MIL: 20 μL; total solution volume:2 mL; pH 8. ^(b)Conditions: Manual agitation time: 30 s; all otherexperimental parameters unchanged. ^(c)Refers to duration of appliedmagnetic field.

Effect of MIL Volume on Extraction Efficiency

The effect of MIL volume on extraction efficiency was investigated forthe [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] and [P_(6,6,6,14+)][FeCl₄—]MILs. A 2 mL solution of 4.17 nM stDNA was extracted using MIL volumesranging from 10 to 25 μL. As shown in FIG. 7, larger volumes ofextraction solvent provided improved DNA extraction efficiencies forboth MILs. Higher extraction efficiencies were obtained using themonocationic [P_(6,6,6,14+)][FeCl₄—] MIL compared to the dicationic[(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] MIL, even at larger droplet volumes.A significant increase in extraction efficiency was observed for the[(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] MIL when the MIL microdroplet volumewas increased from 15 to 20 μL, showing a saturation effect at lowervolumes of extraction solvent. However, the enhancement of extractionefficiency for the [P_(6,6,6,14+)][FeCl₄] MIL was much less pronounced.Because 20 and 25 μL showed similar extraction efficiencies, the smallermicrodroplet volume was used in subsequent tests. All three MILsexamined retained their hydrophobic character and exhibited phaseseparation when subjected to the external magnetic field, even at verylow microdroplet volumes (e.g., 10 μL).

Effect of pH on Extraction Efficiency

The pH of environmental or biological DNA sample solutions is oftenvariable and may have implications on the extraction behavior ofinterfering matrix components. As pH adjustments are often employed insample pretreatment steps to minimize the coextraction of contaminants,it is desired to examine its effect on the MIL-based extraction of DNA.To investigate the effect of pH on extraction efficiency, solutions ofstDNA ranging from pH 2.5-10.9 were prepared and subjected to MIL-basedDDE. The phosphate groups of DNA molecules possess pKa values below thestudied pH range. Therefore, they bear negative charges capable offavorable electrostatic interactions with the MIL cation.

As shown in FIG. 8, the [P_(6,6,6,14+)][FeCl₄] MIL exhibited extractionefficiencies greater than 87% across the pH range studied. Furthermore,the extraction efficiency of stDNA for the [P_(6,6,6,14+)][FeCl₄] MILshowed little dependence on the pH of the solution. In contrast, aconsiderable decrease in extraction efficiency was observed when the[(C₂₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] MIL was used to extract stDNA fromincreasingly basic solutions. In order to maintain high extractionefficiency while avoiding the harsh pH extremes that may compromise thestructural integrity of DNA, pH 8 was selected for subsequentextractions.

Extraction of Single-Stranded Oligonucleotides and Duplex DNA

Short length nucleic acids play a central role in molecular recognitionand hybridization applications. To determine the usefulness ofextracting smaller DNA molecules, MIL-based extraction was applied toshort length single-stranded oligonucleotides and duplex DNA. As shownin Table 3, the extraction of low molecular weight dsDNA and ssDNA isMIL-dependent. In the case of the [(C₈)₃BnN+][FeCl₃Br—] MIL, extractionefficiencies of 69.3% and 57.6% were observed for 20 bp DNA and 33-merssDNA, respectively. However, the same MIL produced an extractionefficiency of only 41.0% for stDNA indicating that it appears topreferentially extract smaller oligonucleotides. In contrast, thedicationic MIL exhibited higher extraction efficiency values for stDNAthan the 20 bp dsDNA, while the [P_(6,6,6,14+)][FeCl₄—] MIL providedextraction efficiencies exceeding 91% for stDNA, 20 bp dsDNA, and ssDNA.These data show that MILs can be designed that are selective forparticular sizes of oligonucleotides or duplex DNA.

TABLE 3 Extraction Efficiencies of dsDNA and ssDNA Using the ThreeHydrophobic MILs % extraction % extraction % extraction efficiencyefficiency efficiency of 20 kbp of 20 bp of 33-mer stDNAa dsDNA^(b)ssDNA^(c) MIL (n = 3) (n = 3) (n = 3) [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2−, 76.8 ±2.6 64.0 ± 1.1 67.7 ± 3.0 FeCl₃Br−] (13) [(C₈)₃BnN+][FeCl₃Br−] (14) 41.0± 0.9 69.3 ± 4.4 57.6 ± 5.0 [P_(6,6,6,14+)][FeCl⁴⁻] 93.8 ± 0.6 91.4 ±0.3 94.0 ± 0.2 aConditions: 4.17 nM; total solution volume: 2 mL; pH 8;volume of MIL 20 μL; manual agitation time: 30 s. ^(b)Conditions: 1224nM; other conditions held constant. ^(c)Conditions: 1499 nM; otherconditions held constant.

Extraction of DNA from a Complex Matrix

The effect of biologically relevant impurities on MIL-based DNAextraction was examined A complex matrix was simulated through theaddition of metal ions or proteins (albumin) to an aqueous solution ofDNA.

The extraction performance of the [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—],[(C₈)₃BnN+][FeCl₃Br—], and [P_(6,6,6,14+)][FeCl₄—] MILs was evaluatedfor 20 kbp stDNA in the presence of NaCl, KCl, CaCl₂.2H₂O, andMgCl₂.6H₂O. FIG. 9 shows that the extraction efficiency for thedicationic ([C₁₆BnIM)₂C₁₂ ²+][NTf2-, FeCl₃Br—] MIL was somewhatdiminished by the addition of the mono- and divalent metal ions, incontrast to what was observed for monocationic imidazolium-based ILs. Avery small to negligible variation in extraction efficiencies wasobserved for the [(C₈)₃BnN+][FeCl₃Br—] and [P_(6,6,6,14+)][FeCl₄—] MILs.

The effect of protein on the extraction efficiency of DNA was done bypreparing aqueous 20 kbp stDNA solutions containing albumin as a modelprotein. The extraction efficiencies of both stDNA and albumin weremonitored over a pH range from 3.5 to 8. As shown in FIGS. 10A-10C, eachof the three MILs exhibited unique extraction behavior in the presenceof stDNA and albumin.

FIG. 10A shows that high extraction efficiencies for both stDNA andalbumin were obtained using the dicationic [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-,FeCl₃Br—] MIL at pH 8. Interestingly, a comparison of FIG. 8 and FIG.10A reveals that the extraction efficiencies of stDNA in the absence ofalbumin were similar to those observed after albumin had been spikedinto the aqueous solution. However, FIG. 8 and FIG. 10B show that theextraction efficiency of stDNA for the [P_(6,6,6,14+)][FeCl₄—] MIL wasdecreased by 46% in the presence of albumin at pH 8. As shown in FIG.10C, the [(C₈)₃BnN+][FeCl₃Br—] MIL provided relatively lower extractionefficiencies of stDNA across the pH range studied.

With an isoelectric point of 4.6, albumin possesses an overall negativecharge at higher pH and may compete with DNA by also engaging inelectrostatic interactions with the MIL cation. To examine this effect,the pH of the sample solution was lowered which resulted in acorresponding decrease in the amount of extracted albumin for the[P_(6,6,6,14+)][FeCl₄—] and [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] MILs.Furthermore, lowering of the sample pH significantly enhanced theextraction efficiency of stDNA for the [P_(6,6,6,14+)][FeCl₄—] MIL.Although these results show that electrostatic interactions between theMIL and albumin are diminished at low pH, coextraction of albumin wasstill observed for all three MILs investigated. While not wishing to bebound by theory, it is now believed this may be due to interactionsbetween the hydrophobic amino acid side chains of albumin and the longalkyl groups of the MIL cations that promote the extraction of protein,regardless of solution pH. As shown in FIG. 10C, the coextraction ofalbumin was less pronounced when employing the [(C₈)₃BnN+][FeCl₃Br—]MIL. Although it extracted less stDNA compared to the other two MILs,the [(C₈)₃BnN+][FeCl₃Br—] MIL exhibited an albumin extraction efficiencyof just 5.0% at pH 4.4, while the [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—]and [P_(6,6,6,14+)][FeCl4-] MILs produced extraction efficienciesnearing 40% at the same pH. These data show that DNA extracted by the[(C₈)₃BnN+][FeCl₃Br—] MIL microdroplet may have less proteincontamination than DNA extracted by the other two MILs under the sameconditions. The extraction behavior of these MILs shows that MIL-basedsolvents are capable of enhancing the selectivity toward DNA in thepresence of proteins.

Recovery of DNA from the MIL Extraction Phase

The recovery of high quality DNA following an extraction step isimportant for accurate downstream analysis, especially in PCR and DNAsequencing experiments. To ensure that DNA extraction performed by theMIL solvent did not alter any portion of the DNA sequence, pDNAextracted by the [(C₈)₃BnN+][FeCl₃Br—] MIL was subjected to sequenceanalysis. The MTAP gene sequence obtained from pDNA extracted by the MILand the sequence of a pDNA standard are shown in FIG. 11 and FIG. 12,respectively. The pDNA extracted by the MIL was shown to contain a MTAPgene identical to the standard, indicating that the pDNA was not alteredduring the MIL extraction step or that the amount of any alterations tothe integrity of the biomolecule are sufficiently low to be detected.

To assess the total quantity of DNA recovered after MIL-based DNAextraction, a 4.17 nM solution of stDNA was extracted using the[(C₈)₃BnN+][FeCl₃Br—] MIL. After dissolution of the stDNA-enriched MILmicrodroplet in 3 M potassium acetate (pH 4.8), the sample was loadedonto silica sorbent. The sorbent was flushed with 1 mL of isopropanol,and the first fraction was collected, which contained stDNA and excesssalt. The stDNA was precipitated with cold ethanol, and the excess saltwas removed by washing the pellet with 80% ethanol. In this approach,HPLC analysis determined the recovery of stDNA from the MIL microdropletto be 57±6%. The yield of the MIL-based DDE method was 23.5 μg of stDNA.Comparatively, a QlAamp DNA Mini Kit was capable of recovering 84±5% ofthe stDNA from a 4.17 nM solution with a yield of 84.4 μg.

Extraction of DNA from Bacterial Cell Lysate

To show the applicability of the MIL-based DNA extraction method, pDNAin an E. coli cell lysate was extracted using the [(C₈)₃BnN+][FeCl₃Br—]MIL and subjected to PCR. This MIL was chosen to minimize proteincoextraction. The following two methods were employed for the isolationof DNA from the MIL extraction phase: an approach targeting greaterquantities of high purity DNA and a rapid approach for recovering asufficient quantity of high quality template DNA for PCR. In order toassess whether each recovery procedure was capable of isolatingPCR-amplifiable DNA from E. coli, pDNA was extracted from a bacterialcell lysate using the [(C₈)₃BnN+][FeCl₃Br—] MIL and subjected to boththe silica-based and the rapid immersion method.

As shown in FIG. 13, the silica-based method provided a more intense PCRproduct band (Lane 3) than did the rapid immersion approach (Lane 2).Nonetheless, immersion of the pDNA-enriched MIL microdroplet in Tris-HClfor just 2 min was capable of transferring sufficient pDNA for PCRamplification and visual detection of the MTAP gene on an agarose gel.This method is especially useful for high throughput nucleic acidanalyses, such as the rapid screening of an environmental sample formicroorganisms or identification of DNA biomarkers in virtually anysample.

Discussion of Example 2

Hydrophobic MILs were employed as solvents for the extraction of DNAfrom aqueous solution. The MIL-based method allows for rapid, highlyefficient extractions providing a DNA-enriched microdroplet that iseasily manipulated in aqueous solution by application of a magneticfield. Higher extraction efficiencies were obtained for shorteroligonucleotides and DNA duplexes with the [(C₈)₃BnN+][FeCl₃Br—] MIL,while the dicationic [(C₁₆BnIM)₂C₁₂ ²⁺][NTf2-, FeCl₃Br—] MIL affordedhigher extraction efficiencies for the much longer stDNA. MIL-basedextraction of stDNA from a complex matrix containing albumin furthershow the desirable extraction profiles for the MILs, revealingcompetitive extraction behavior for the [P_(6,6,6,14+)][FeCl₄—] MIL andless pronounced coextraction for the [(C₈)₃BnN+][FeCl₃Br—] MIL. Theseresults show how the structural customization of MILs is useful toachieve enhanced selectivity toward a variety of DNA samples. Therecovery of DNA from the MIL extraction phase which was determined to be57±6%, is also shown herein. Furthermore, sequence analysis demonstratedthat the DNA recovered from the MIL extraction phase was intact and thesequence unmodified. Plasmid DNA from a bacterial cell lysate wasextracted using MIL-based DDE and shown to provide sufficient pDNAquantity and quality for PCR. Further, these MILs are especially usefulas solvent systems in many other applications. One non-limitingapplication is in microfluidic devices where their paramagneticproperties can be exploited for precise control of sample movement.

Example 3 Analysis of Bacterial Plasmid DNA by Solid-Phase MicroExtraction

A cross-linked PIL sorbent coating consisting of a dicationic ILbasedcross-linker 1,12-bis(3-vinylimidazolium) dodecane dibromide ([VIM)₂C₁₂²⁺]2[Br⁻]) and 1-vinyl-3-hexylimidazolium chloride ([VHIM⁺] [Cl⁻])monomer was used for the extraction and preconcentration of plasmid DNA(pDNA) from bacterial cells. The PIL-based SPME method was capable ofextracting sufficient template pDNA after 5 min from a 20 ng/mL solutionfor subsequent PCR amplification and visualization on an agarose gel.Due to favorable electrostatic interactions afforded by the PIL-basedsorbent coating, a greater quantity of template pDNA was extracted fromaqueous solution compared to a commercial polyacrylate (PA)-basedsorbent coating under similar extraction conditions. The PIL sorbent wassuccessfully applied for the extraction of two different plasmids from amixture of E. coli transformants.

Reagents

1-vinylimidazole, 1-chlorohexane, 1,12-dibromododecane,2-hydroxy-2-methylpropiophenone (DAROCUR 1173), andvinyltrimethoxysilane (VTMS) were purchased from Sigma-Aldrich(Milwaukee, Wis., USA). Methanol, concentrated hydrogen peroxide (30%(w/w)), hydrochloric acid, sodium chloride,tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), acetic acid,boric acid, phosphoric acid, and sodium hydroxide were obtained fromFisher Scientific (Fair Lawn, N.J., USA). Deionized water (18.2 MΩ cm)was obtained from a Milli-Q water purification system (Millipore,Bedford, Mass., USA). Elastic nitinol wires with an outer diameter of125 μm were purchased from Nitinol Devices & Components (Fremont,Calif., USA). A UV reactor equipped with a spinning carousel wasobtained from Southern New England Ultraviolet Company (Bradford, Conn.,USA). Amber glass vials (10 mL), screw caps with polytetrafluoroethylenesepta, and an 85 μm polyacrylate (PA) fiber were obtained from Supelco(Bellefonte, Pa., USA). Polypropylene microcentrifuge tubes werepurchased from Fischer Scientific. Modified pET-32 plasmids wereobtained from EMD Millipore (Billerica, Mass., USA). NEB 5-alphaCompetent E. coli cells and Phusion High-Fidelity DNA Polymerase and 5×Phusion HF buffer were obtained from New England Biolabs (Ipswich,Mass., USA). A deoxyribonucleoside triphosphate (dNTP) mix (10 mM each)was obtained from Thermo Scientific (Pittsburgh, Pa., USA). Agarose andtris(hydroxymethyl)aminomethane (Tris) were purchased from P212121(Ypsilanti, Mich., USA). A 1 Kb Plus DNA Ladder (250-25,000 bp) wasobtained from Gold Biotechnology, Inc. (St. Louis Mo., USA). SYBR® SafeDNA gel stain was supplied by Life Technologies (Carlsbad, Calif., USA)and bromophenol blue was purchased from Santa Cruz Biotech (Dallas,Tex., USA).

Preparation of PIL-Based SPME Fiber

The IL monomer ([VHIM⁺][Cl⁻]) and the dicationic IL cross-linker([(VIM)₂C₁₂ ²⁺] 2[Br⁻]) were synthesized. Cross-linked PIL-based SPMEfibers were prepared. Briefly, the nitinol support was immersed inboiling hydrogen peroxide at 70-75° C. for 2 h to generate free hydroxylgroups on the surface of the substrate. The derivatized surface was thenreacted with neat VTMS at 85° C. for 2 h. A 1 cm length of the metallicalloy fiber was then dip-coated with a mixture containing the ILmonomer, 50% (w/w) cross-linker with respect to monomer, and 3% (w/w)DAROCUR 1173. Afterwards, the fiber was exposed to 254 nm UV light for 2h. The PIL-based SPME fiber was then immersed in 100 μL of methanol for15 mM, followed by washing in 10 mL of 20 mM Tris-HCl for 30 mM. A JEOLJSM-7500F scanning electron microscope (SEM) was used to characterizethe PIL-based SPME fiber. A SEM image of a PIL-based fiber after 40extractions is shown in FIG. 14. The average film thickness of the PILsorbent coating was found to be approximately 65 μm, which is slightlyless than the film thickness of the commercial PA fiber (85 μm) examinedin this example.

Cell Cultures

Competent E. coli cells were transformed with a modified pET-32 plasmid(5.9 kbp) containing either the 879 bp human 5′-methylthioadenosinephosphorylase (MTAP) gene or the 1275 bp vaccinia virus K4 gene. Thetransformed cells were cultured in 120 mL of Luria Bertani (LB) mediawith 100 μg/mL carbenicillin at 37° C. for 20 h. Optical densitymeasurements were performed at 600 nm using a Nicolet Evolution 300UV-vis spectrophotometer from Thermo Scientific. Purified pDNA wasobtained from the culture with a QlAprep Spin Miniprep Kit (Qiagen,Valencia, Calif., USA) according to the manufacturer's instructions. Theconcentration of the pDNA standards were measured using a Synergy H4Hybrid Microplate Reader from BioTek (Winooski, Vt., USA).

PIL-Based SPME of pDNA

The buffer was prepared using a mixture of 0.04 M of boric acid,phosphoric acid, and acetic acid. The desired pH was obtained byadjusting with sodium hydroxide or hydrochloric acid. Solutionscontaining either E. coli cells or purified pDNA were prepared in 10 mLof the buffer immediately prior to PIL-based SPME. The number of cellsadded to solution was approximated using OD₆₀₀ values. pDNA wasextracted by immersing the SPME fiber in the solution under agitation at650 rpm. Following extraction, the pDNA was desorbed by placing the PILfiber in a solution containing 50 μL of 1 M NaCl and 20 mM Tris-HCl for15 mM. A 2 μL aliquot of the desorption solution was then subjected toPCR amplification. The PIL fiber was washed using 2×10 mL of 1 M NaCl inthe buffer for 5 mM before further use.

PCR and Amplicon Visualization

Immediately prior to PCR amplification, 0.03% (w/v) of Brij-700 wasadded to the desorption solution and incubated at room temperature for15 mM with gentle mixing to prevent adsorption of the nucleic acid tothe polypropylene tube. A 2 μL aliquot of the desorption solution wasthen transferred to a PCR tube containing 34.5 μL of deionized water and10 μL of 5× Phusion HF buffer. The MTAP and K4 genes were amplifiedusing the primers 5′-TGC TGT TCC AGG GAC CT-3′ [SEQ ID NO:3] and 5′-GAATTC GGA TCC GGA CGC-3′ [SEQ ID NO:4] at a final concentration of 0.2 μM.

Finally, 1 μL from a 10 mM stock solution of dNTPs was added to thereaction mixture along with 1 unit of Phusion High Fidelity DNAPolymerase. Amplification of the MTAP and K4 genes was performed using aTechne FTgene2D thermal cycler (Burlington, N.J., USA). Thermalconditions for the amplification of both genes consisted of 5 mM initialdenaturation at 95° C. followed by 30 cycles of denaturation (95° C. for30 s), annealing (54° C. for 45 s), and elongation (72° C. for 45 s).

PCR products were loaded onto a 1% agarose gel and separated using a BRLH4 Horizontal Gel Electrophoresis System from Life Technologies with aNeo/Sci dual output power supply (Rochester, N.Y., USA). Gels werevisualized on a Safe Imager™ 2.0 blue-light transilluminator from LifeTechnologies. The intensities of the DNA bands were measured usingImageJ software (National Institutes of Health).

Preconcentration of pDNA Using SPME Fibers

The analysis of extremely small quantities of DNA invariably requiressample purification and preconcentration prior to downstream procedures.Conventional DNA sample preparation methods involve numerous steps thatare time consuming and often require manual operation. The developmentof SPME sorbent coatings for DNA sample preparation is an importantadvancement that not only facilitates preconcentration, but alsoconstitutes a platform that is amenable to laboratory, clinical, orfield sampling.

pDNA containing the MTAP gene (879 bp) was chosen as a model DNAbiomolecule to investigate the extraction performance of thecross-linked PIL and commercial PA SPME fibers. FIG. 15 shows the PCRamplification of the MTAP gene from pDNA extracted from aqueous solutionusing PIL and PA SPME fibers. As shown in Lane 1, the amplicon is notobserved after directly loading 2 μL of the 20 ng/mL pDNA solution ontothe gel. This is attributed to an insufficient amount of template todetect the amplicon after 30 cycles of PCR amplification.

In contrast, preconcentration of pDNA by the PIL or PA fiber providessufficient template DNA for detection of the amplicon in Lanes 2 and 3,respectively. The intensity of the PCR product band obtained after PILextraction was calculated to be 4.2 times greater than the band observedfor the PA fiber indicating that a greater quantity of pDNA wasextracted by the PIL coating. Previously, it was believed that IL-basedsubstrates are capable of engaging in electrostatic interactions withthe negatively charged phosphate backbone of DNA molecules. Unlike thePA sorbent coating, however, it is now shown herein that thecross-linked PIL coating possesses ion-exchange sites that favorelectrostatic interactions and lead to superior enrichment of pDNA.

Optimization of Extraction and Desorption Conditions

The nature of the desorption solution has a profound influence on therecovery of pDNA from the PIL-based SPME fiber. As shown in FIG. 16,desorption of the PIL fiber in a solution of 20 mM Tris-HCl afterextraction from a 20 ng/mL pDNA solution resulted in no observableamplicon. However, the addition of 1 M NaCl to the desorption solutionprovided a dramatic improvement in the recovery of pDNA from the sorbentcoating. At NaCl concentrations greater than 1 M, inhibition of PCR wasobserved. In an effort to maximize the recovery of pDNA from the PILcoating while maintaining PCR amplification, 1 M NaCl was used as thedesorption solution in all subsequent experiments.

To avoid carryover of pDNA, the PIL-based SPME fiber was washed aftereach extraction using 2×10 mL of 1 M NaCl in buffer for 5 min. The PILfiber was then desorbed in 50 μL of 1 M NaCl for 15 min and a 2 μLaliquot of the desorption solution was subjected to PCR. After washingthe PIL sorbent, no amplicon band was observed.

An aqueous solution containing 20 ng/mL of pDNA was used to determinethe effect of extraction time on PIL-based SPME. The extraction time wasvaried from 5 to 30 min while the desorption time was held constant at15 min.

As shown in FIG. 17, an increasing amount of amplicon was obtained asthe extraction time was increased. However, beyond an extraction time of30 min, no significant increase in the amount of pDNA extracted wasobserved. An extraction time of 20 min was selected as a compromisebetween extraction time and the amount of pDNA extracted.

The effect of solution pH on the extraction of pDNA using the PIL-basedsorbent coating was studied. Aqueous solutions containing 20 ng/mL ofpDNA and pH values ranging from 2 to 8 were examined. As shown in FIG.18, modest differences in the amount of amplicon were observed from pH 4to 8 with a slightly higher amount of pDNA being extracted at pH 4.0. Incontrast, extraction at pH 2.0 generated the lowest intensity ampliconband, most likely due to degradation of the template pDNA.

In order to ensure that the sequence of the pDNA remained unalteredfollowing extraction by the PIL coating, the amplified MTAP gene wassubjected to sequence analysis. The sequence of the PCR amplified MTAPgene obtained after PIL-based SPME and the sequence of a MTAP genestandard are shown in FIG. 19 and FIG. 20. Comparison of the MTAP genesequence after PIL-based extraction to the standard revealed nodetectable differences, demonstrating the feasibility of PIL-based SPMEas a DNA sample preparation technique.

Direct Extraction of pDNA from a Bacterial Cell Culture

The sampling of bacterial DNA from environmental, food, and biologicalsamples is a vital step for the identification of microbial communitiesand pathogen detection. Frequently, the population of microorganisms ina given sample is diverse. To determine whether the PIL-based SPMEmethod is capable of detecting bacteria from independent cell cultures,E. coli cells transformed with either the MTAP or K4 plasmids were mixedin a 1:1 ratio and diluted to 10 mL with buffer. The total number ofcells in solution containing the MTAP plasmid was calculated to be1.44×10⁸, while 1.35×10⁸ cells contained the K4 plasmid. FIG. 21 showsthe PCR products obtained from the desorption solution followingPIL-based SPME of the diluted bacterial cells. The bands for theamplified MTAP and K4 genes are of similar intensity, indicating thatthe length of the gene insert does not affect the relative proportionsof pDNA extracted.

A recurrent cause of bias in the analysis of microbial communitiesoccurs during sampling procedures as a result of disproportionatequantities of unique specimens in the sample. A 10 mL aqueous solutionof E. coli transformants possessing either the K4 gene or the MTAP gene(100:1, respectively) was extracted using the PIL sorbent coating. Thenumber of E. coli cells in solution containing the K4 and MTAP genes was1.36×10⁸ and 1.44×10⁶, respectively. As shown in FIG. 22, bands for boththe MTAP and K4 genes were observed following PCR amplification.Normalizing the band intensity for the K4 gene to 1, the MTAP geneproduced a band with an intensity of 0.086, reflecting the 100-foldfewer E. coli cells possessing the MTAP gene in solution.

Thus, the PIL-based SPME method is especially useful for the rapidanalysis of bacterial contamination in food samples or the determinationof microbial diversity in environmental samples. Moreover, the SPMEplatform is well suited for applications requiring field sampling,further expanding the applicability of the method.

Discussion of Example 3

PIL-based sorbent coatings were used in SPME for the isolation andpreconcentration of pDNA from bacterial cells. The PIL sorbent phaseexhibited superior extraction of pDNA from aqueous solution compared toa commercial PA sorbent coating. The optimized SPME technique wascapable of preconcentrating sufficient pDNA within 5 min for PCRamplification and detection on an agarose gel. Sequence analysis of atarget gene from the extracted pDNA confirmed that the integrity of thepDNA sequence was preserved after PIL-based SPME. The developed methodwas successfully employed for the analysis of two different E. colitransformants from a dilute solution.

While the materials and methods have been described with reference tovarious and preferred embodiments, it should be understood by thoseskilled in the art that various changes can be made and equivalents canbe substituted for elements thereof without departing from the essentialscope of the invention. In addition, many modifications can be made toadapt a particular situation or material to the teachings of theinvention without departing from the essential scope thereof.

Therefore, it is intended that the invention not be limited to theparticular embodiment disclosed herein contemplated for carrying outthis invention, but that the invention will include all embodimentsfalling within the scope of the claims.

The publication and other material used herein to illuminate theinvention or provide additional details respecting the practice of theinvention, are incorporated by reference herein, and for convenience areprovided in the following bibliography.

Citation of the any of the documents recited herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicant anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

What is claimed is:
 1. A magnetic ionic liquid, comprising at least one cationic component and at least one anionic component, wherein at least one of the cationic components or at least one of the anionic components is a paramagnetic component, the magnetic ionic liquid being capable of manipulation by an external magnetic field.
 2. The magnetic ionic liquid of claim 1, wherein the cationic component is monocationic, dicationic or tricationic.
 3. The magnetic ionic liquid of claim 1, wherein the cationic component is an asymmetric cationic component.
 4. The magnetic ionic liquid of claim 2, wherein the magnetic ionic liquid comprises a monocationic compound selected from:

wherein R is selected from the group consisting of:


5. The magnetic ionic liquid of claim 2, wherein the magnetic ionic liquid comprises a dicationic compound selected from:

wherein R is selected from the group consisting of: C₁₆H₃₃ (6) and

and,


6. The magnetic ionic liquid of claim 2, wherein the magnetic ionic liquid comprises a tricationic compound selected from:


7. The magnetic ionic liquid of claim 2, wherein the magnetic ionic liquid comprises a compound selected from of:


8. The magnetic ionic liquid of claim 1, comprising multiple magnetic iron(III) centers.
 9. The magnetic ionic liquid of claim 1, wherein the paramagnetic component comprises a high spin transition metal.
 10. The magnetic ionic liquid of claim 1, wherein the paramagnetic component comprises a high-spin d⁵ iron(III) center.
 11. The magnetic ionic liquid of claim 1, comprising one or more of: benzyl substituents; dysprosium; a benzimidazolium cation; an imidazolium cation.
 12. The magnetic ionic liquid of claim 1, wherein the anionic component comprises three anions.
 13. The magnetic ionic liquid of claim 1, wherein the anionic component is selected from: a [FeCl₃Br⁻] anion and a [NTf₂ ⁻] anion.
 14. The magnetic ionic liquid of claim 1, wherein the ionic liquid has an effective magnetic moment of up to 11.76 Bohr magnetons.
 15. A method of increasing the effective magnetic moment of an ionic liquid of claim 1, comprising: incorporating an additional iron(III) center into an ionic liquid, and thereby increasing the effective magnetic moment of the ionic liquid; by using one of: the steps shown in Scheme 1a or Scheme 1b; the steps shown in Scheme 2, the steps shown in Scheme 3, or the steps shown in Scheme
 4. 16. A method of conducting gas chromatography, comprising using a magnetic ionic liquid of claim 1 as a stationary phase.
 17. A method of conducting nucleic acid extraction, comprising using a magnetic ionic liquid of claim 1 as a solvent to extract a nucleic acid from an aqueous solution; and, optionally, subjecting the extracted nucleic acid to a polymerase chain reaction (PCR) process. 